Production of proteins in glutamine-free cell culture media

ABSTRACT

The present invention relates generally to glutamine-free cell culture media supplemented with asparagine. The invention further concerns the production of recombinant proteins, such as antibodies, in asparagine-supplemented glutamine-free mammalian cell culture.

RELATED APPLICATIONS

This application claims the benefit under 35 USC §119 to U.S.Provisional Application 61/232,889 filed Aug. 11, 2009.

FIELD OF THE INVENTION

The present invention relates generally to glutamine-free cell culturemedia. The invention further concerns the production of recombinantproteins, such as antibodies, in glutamine-free mammalian cell culture.

BACKGROUND OF THE INVENTION

Mammalian cells have become the dominant system for the production ofmammalian proteins for clinical applications, primarily due to theirability to produce properly folded and assembled heterologous proteins,and their capacity for post-translational modifications. It isconventional to have glutamine in cell culture media during recombinantproduction of heterologous proteins, including antibodies. L-glutamineis an essential amino acid, which is considered the primary energy andnitrogen sources for cells in culture. Most commercially available mediaare formulated with free L-glutamine which is either included in thebasal formula or added to liquid media formulations at the time of use.Thus, all mammalian cell culture media contain glutamine except thosefor glutamine synthetase transfected cell lines, such as GS NS0 and GSCHO cell lines, where the cells themselves produce the glutamine neededfor growth. Glutamine is widely used at various concentrations typicallyfrom 1 to 20 mM in base media and much higher concentration in feeds forfed-batch process. For example, the concentration of L-glutamine is 0.5mM in Ames' Medium and 10 mM in MCDP Media 131. DMEM/Ham's NutrientMixture F-12 (50:50) is often used as a starting formulation forproprietary media used with Chinese Hamster Ovary (CHO) cells.L-glutamine in DMEM/Ham's Nutrient Mixture F-12 is 2.5 mM. L-glutamineconcentration in Serum-Free/Protein Free Hybridoma Medium is 2.7 mM.L-glutamine in DMEM, GMEM, IMDM and H-Y medium is 4 mM, of which IMDM isoften used as a starting formulation for proprietary hybridoma cellculture media. It is generally held that hybridoma cells grow better inconcentrations of L-glutamine that are above the average levels found inmedia. (Dennis R. Conrad, Glutamine in Cell Culture, Sigma-Aldrich MediaExpert)

It was shown that glutamine is the main source of ammonia accumulated incell culture (see review by Markus Schneider, et. al. 1996, Journal ofBiotechnology 46:161-185). Thus, lowering glutamine in cell culturemedia significantly reduced the accumulation of NH₄ ⁺ level, resultingin lower cytotoxicity (see Markus Schneider, et. al. 1996, supra).Reduced NH₄ ⁺ cytotoxicity resulted in higher cell viability, thusextended culture longevity. Based on an estimated glutamine consumptionstudy using CHO cells, it was suggested that cells may consume glutamineat a rate of 0.3-0.4 mM per day (Miller, et. al. 1988, Biotechnol.Bioeng. 32: 947-965). Altamirano et al. (2001, J. Biotechnol. 110:171-9)studied the effect of glutamine replacement by glutamate and the balancebetween glutamate and glucose metabolism on the redistribution of CHOcells producing recombinant human tissue plasminogen activator(rhut-PA). When glutamine was replaced with glutamate and balanced withglucose catabolism (carbon and nitrogen ratio, C/N ratio), cellmetabolism was found redistributed and forced to utilize carbon andenergy source more favorably to production of rhut-PA. It was alsoreported that CHO cells in adherent cultures can grow in the absence ofadded glutamine due to endogenous glutamine synthetase activity thatallowed cells to synthesize glutamine from glutamic acid in the medium(Sanfeliu and Stephanopoulos, 1999, Biotechnol. Bioeng. 64:46-53).However, compared to control cultures in glutamine-containing media, thecell growth rate in glutamine-free media was slower with an increasedfraction of cells distributed in the G0/G1 phase. The depletion of bothglutamine and glutamic acid did cause cell death.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the unexpectedfinding that not only can recombinant proteins be produced in amammalian host cell using a glutamine-free production medium without anysignificant adverse effect, in fact the use of a glutamine-free mediumin the production phase significantly increases cell viability, culturelongevity, specific productivity and/or the final recombinant proteintiter.

The present invention is also based on the unexpected finding that theaddition of asparagine to a glutamine-free production medium can furtherenhance the cell viability, culture longevity, specific productivityand/or the final recombinant protein titer in a mammalian host cellusing a glutamine-free production medium without any significant adverseeffect.

In one aspect, the invention concerns a process for producing apolypeptide in a mammalian host cell expressing said polypeptide,comprising culturing the mammalian host cell in a production phase ofthe culture in a glutamine-free production culture medium supplementedwith asparagine.

In one embodiment, the mammalian host cell is a Chinese Hamster Ovary(CHO) cell.

In another embodiment, the mammalian host cell is a dhfr⁻ CHO cell.

In yet another embodiment, the production medium is serum-free.

In a further embodiment, the production culture medium comprises one ormore ingredients selected from the group consisting of

1) an energy source;

2) essential amino acids;

3) vitamins;

4) free fatty acids; and

5) trace elements.

In a still further embodiment, wherein the production culture mediumadditionally comprises one or more ingredients selected from the groupconsisting of:

1) hormones and other growth factors;

2) salts and buffers; and

3) nucleosides.

In all embodiments, the production phase may, for example, be a batch orfed batch culture phase.

In all embodiments, the process may further comprise the step ofisolating said polypeptide.

In a further embodiment, isolation may be followed by determining one ormore of cell viability, culture longevity, specific productivity andfinal recombinant protein titer following isolation.

In a still further embodiment, at least one of the cell viability,culture longevity, specific productivity and final recombinant proteintiter is increased relative to the same polypeptide produced in aglutamine-containing production medium of the same composition.

In a further aspect, the invention concerns a ready-to-useglutamine-free cell culture medium for the production of a polypeptidein a production phase.

In yet another embodiment, the polypeptide is a mammalian glycoprotein.

In other embodiments, the polypeptide is selected from the groupconsisting of antibodies, antibody fragments, and immunoadhesins.

In all embodiments, the polypeptide may, for example, be an antibody, ora biologically functional fragment of an antibody. Representativeantibody fragments include Fab, Fab′, F(ab′)₂, scFv, (scFv)₂, dAb,complementarity determining region (CDR) fragments, linear antibodies,single-chain antibody molecules, minibodies, diabodies, andmultispecific antibodies formed from antibody fragments.

In a still further embodiment, the antibody or antibody fragment ischimeric, humanized or human.

Therapeutic antibodies include, without limitation, anti-HER2 antibodiesanti-CD20 antibodies; anti-IL-8 antibodies; anti-VEGF antibodies;anti-CD40 antibodies, anti-CD11a antibodies; anti-CD18 antibodies;anti-IgE antibodies; anti-Apo-2 receptor antibodies; anti-Tissue Factor(TF) antibodies; anti-human a43₇ integrin antibodies; anti-EGFRantibodies; anti-CD3 antibodies; anti-CD25 antibodies; anti-CD4antibodies; anti-CD52 antibodies; anti-Fc receptor antibodies;anti-carcinoembryonic antigen (CEA) antibodies; antibodies directedagainst breast epithelial cells; antibodies that bind to colon carcinomacells; anti-CD38 antibodies; anti-CD33 antibodies; anti-CD22 antibodies;anti-EpCAM antibodies; anti-GpIIb/IIIa antibodies; anti-RSV antibodies;anti-CMV antibodies; anti-HIV antibodies; anti-hepatitis antibodies;anti-CA 125 antibodies; anti-αvβ3 antibodies; anti-human renal cellcarcinoma antibodies; anti-human 17-1A antibodies; anti-human colorectaltumor antibodies; anti-human melanoma antibody R24 directed against GD3ganglioside; anti-human squamous-cell carcinoma; and anti-humanleukocyte antigen (HLA) antibodies, and anti-HLA DR antibodies.

In other embodiments, the therapeutic antibody is an antibody binding toa HER receptor, VEGF, IgE, CD20, CD11a, CD40, or DR5.

In other embodiments, the therapeutic antibody is an anti-BR3 antibodyor BR3-Fc immunoadhesin.

In other embodiments of the method of the present invention, thepolypeptide expressed in the recombinant host cell is a therapeuticpolypeptide. For example, the therapeutic polypeptide can be selectedfrom the group consisting of a growth hormone, including human growthhormone and bovine growth hormone; growth hormone releasing factor;parathyroid hormone; thyroid stimulating hormone; lipoproteins;alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin;follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon;clotting factors such as factor VIIIC, factor IX, tissue factor, and vonWillebrands factor; anti-clotting factors such as Protein C; atrialnatriuretic factor; lung surfactant; a plasminogen activator, such asurokinase or human urine or tissue-type plasminogen activator (t-PA);bombesin; thrombin; hemopoietic growth factor; tumor necrosisfactor-alpha and -beta; enkephalinase; RANTES (regulated on activationnormally T-cell expressed and secreted); human macrophage inflammatoryprotein (MIP-1-alpha); a serum albumin such as human serum albumin;Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; a microbial protein,such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associatedantigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelialgrowth factor (VEGF); receptors for hormones or growth factors; ProteinA or D; rheumatoid factors; a neurotrophic factor such as bone-derivedneurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4,NT-5, or NT-6), or a nerve growth factor such as NGF-β; platelet-derivedgrowth factor (PDGF); fibroblast growth factor such as aFGF and bFGF;epidermal growth factor (EGF); transforming growth factor (TGF) such asTGF-alpha and TGF-beta, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, orTGF-β5; insulin-like growth factor-I and -II (IGF-I and IGF-II);des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor bindingproteins; CD proteins such as CD3, CD4, CD8, CD19, CD20, CD34, and CD40;erythropoietin; osteoinductive factors; immunotoxins; a bonemorphogenetic protein (BMP); an interferon such as interferon-alpha,-beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF,GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxidedismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor; viral antigen such as, for example, a portion ofthe AIDS envelope; transport proteins; homing receptors; addressins;regulatory proteins; integrins such as CD11a, CD11b, CD11c, CD 18, anICAM, VLA-4 and VCAM; a tumor associated antigen such as HER2, HER3 orHER4 receptor; and fragments of said polypeptides.

In all embodiments, the recombinant host cell can be an eukaryotic hostcell. such as a mammalian host cell, including, for example, ChineseHamster Ovary (CHO) cells.

These and other aspects will be apparent from the description below,including the Examples and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Apomab antibody cube plot analysis of titer results from a FullFactorial Design of Experiment (DOE) evaluating the effect of differentconcentrations of Glutamine, Glutamate, Asparagine and Aspartate. Themodel predicts that the highest titer is achieved in Glutamine-Freemedia supplemented with 10 mM Asparagine, 10 mM Aspartic Acid and 1 mMGlutamic Acid.

FIG. 2. BR3-Fc immunoadhesin cube plot analysis of titer results from aFull Factorial DOE evaluating the effect of different concentrations ofGlutamine, Glutamate, Asparagine and Aspartate. The model predicts thatthe highest titer is achieved in Glutamine-Free media supplemented with10 mM Asparagine, 10 mM Aspartic Acid and 1 mM Glutamic Acid.

FIG. 3. anti-VEGF antibody cube plot analysis of titer results from aFull Factorial DOE evaluating the effect of different concentrations ofGlutamine, Glutamate, Asparagine and Aspartate. The model predicts thatthe highest titer is achieved in Glutamine-Free media supplemented with10 mM Asparagine, 10 mM Aspartic Acid and 1 mM Glutamic Acid.

FIG. 4. Effect of Asparagine under Glutamine-free, low Glutamate andhigh Aspartate conditions on Apomab antibody titer. In Glutamine-freemedium, Apomab antibody titer was significantly increased in thepresence of 2.5-15 mM Asparagine compared to Glutamine-free cultureswithout Asparagine. Under these conditions, the presence or absence ofGlutamate had no effect on titer.

FIG. 5. Apomab antibody titer production across various Asparagine andAspartate concentrations in Glutamine-free and low Glutamate conditions.A positive titration effect was observed when increasing Aspartate from0 to 10 mM under these conditions.

FIGS. 6. A-C. Effect of glutamine-free medium supplemented with 10 mMAsparagine, 10 mM Aspartic Acid and 1 mM Glutamic Acid on titer. Thefinal titer for Apomab antibody, anti-VEGF antibody and BR3-Fcimmunoadhesin was significantly higher in Glutamine-free medium comparedto Glutamine-containing medium.

FIGS. 7A and B. Effect of DMEM/F12 glutamine-free medium supplementedwith 10 mM Asparagine, 10 mM Aspartic Acid and 1 mM Glutamic Acid ontiter. The final titer for Apomab antibody and anti-VEGF antibody wassignificantly higher in Glutamine-free DMEM/F12 medium compared toGlutamine-containing DMEM F12 medium.

FIGS. 8A-C. Effect of glutamine-free medium supplemented with 10 mMAsparagine, 10 mM Aspartic Acid and 1 mM Glutamic Acid on cell specificproductivity (Qp). Cell specific productivity for Apomab antibody,anti-VEGF antibody and BR3-Fc immunoadhesin was significantly higher inGlutamine-free medium compared to Glutamine-containing medium.

FIGS. 9A and B. Effect of DMEM/F12 glutamine-free medium supplementedwith 10 mM Asparagine, 10 mM Aspartic Acid and 1 mM Glutamic Acid oncell specific productivity (Qp). Cell specific productivity for Apomabantibody and anti-VEGF antibody was significantly higher inGlutamine-free DMEM/F12 medium compared to Glutamine-containing DMEM/F12medium.

FIGS. 10A-C. Effect of glutamine-free medium supplemented with 10 mMAsparagine, 10 mM Aspartic Acid and 1 mM Glutamic Acid on CellViability. Cell viability for Apomab antibody, anti-VEGF antibody andBR3-Fc immunoadhesin was higher in Glutamine-free medium compared toGlutamine-containing medium.

FIGS. 11A and B. Effect of DMEM/F12 glutamine-free medium supplementedwith 10 mM Asparagine, 10 mM Aspartic Acid and 1 mM Glutamic Acid onCell Viability. In DMEM/F12 medium, cell viability was not consistentlyimproved in Glutamine-free medium. Viability was higher for Apomabantibody, but lower for anti-VEGF antibody compared to Glutaminecontaining medium.

FIGS. 12 A-C. Effect of glutamine-free medium supplemented with 10 mMAsparagine, 10 mM Aspartic Acid and 1 mM Glutamic Acid on ammoniaformation Ammonia was usually lower in Glutamine-free cultures comparedto Glutamine-containing cultures.

FIGS. 13A and B. Effect of DMEM/F12 glutamine-free medium supplementedwith 10 mM Asparagine, 10 mM Aspartic Acid and 1 mM Glutamic Acid onammonia formation. Ammonia was significantly reduces in Glutamine-freeDMEM/F12 medium compared to Glutamine-containing DMEM/F12 medium.

DETAILED DESCRIPTION OF THE INVENTION A. Definitions

The terms “cell culture medium”, “culture medium”, and “nutrientmixture” refer to a nutrient solution used for growing mammalian cellsthat typically provides at least one component from one or more of thefollowing categories:

1) an energy source, usually in the form of a carbohydrate such asglucose;

2) some or all of the essential amino acids, and usually the basic setof twenty amino acids plus cystine;

3) vitamins and/or other organic compounds typically required at lowconcentrations;

4) free fatty acids; and

5) trace elements, where trace elements are defined as inorganiccompounds or naturally occurring elements that are typically required atvery low concentrations, usually in the micromolar range.

The nutrient mixture may optionally be supplemented with one or morecomponent from any of the following categories:

1) hormones and other growth factors as, for example, insulin,transferrin, and epidermal growth factor;

2) salts and buffers as, for example, calcium, magnesium, and phosphate;and

3) nucleosides such as, for example, adenosine and thymidine.

The cell culture medium is generally “serum free” when the medium isessentially free of serum from any mammalian source (e.g. fetal bovineserum (FBS)). By “essentially free” is meant that the cell culturemedium comprises between about 0-5% serum, preferably between about 0-1%serum, and most preferably between about 0-0.1% serum. Advantageously,serum-free “defined” medium can be used, wherein the identity andconcentration of each of the components in the medium is known (i.e., anundefined component such as bovine pituitary extract (BPE) is notpresent in the culture medium).

In the context of the present invention the expressions “cell”, “cellline”, and “cell culture” are used interchangeably, and all suchdesignations include progeny. Thus, the words “transformants” and“transformed (host) cells” include the primary subject cell and culturesderived therefrom without regard for the number of transfers. It is alsounderstood that all progeny may not be precisely identical in DNAcontent, due to deliberate or inadvertent mutations. Mutant progeny thathave the same function or biological activity as screened for in theoriginally transformed cell are included. Where distinct designationsare intended, it will be clear from the context.

The term “animal host cell,” “animal cell,” “animal recombinant hostcell,” and the like, encompasses invertebrate, non-mammalian vertebrate(e.g., avian, reptile and amphibian) and mammalian cells. Examples ofinvertebrate cells include the following insect cells: Spodopterafrugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus(mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori. See,e.g., Luckow et al., Bio/Technology, 6:47-55 (1988); Miller et al., inGenetic Engineering, Setlow, J. K. et al., eds., Vol. 8 (PlenumPublishing, 1986), pp. 277-279; and Maeda et al., Nature, 315:592-594(1985).

The term “mammalian host cell,” “mammalian cell,” “mammalian recombinanthost cell,” and the like, refer to cell lines derived from mammals thatare capable of growth and survival when placed in either monolayerculture or in suspension culture in a medium containing the appropriatenutrients and growth factors. The necessary nutrients and growth factorsfor a particular cell line are readily determined empirically withoutundue experimentation, as described for example in Mammalian CellCulture (Mather, J. P. ed., Plenum Press, N. Y. (1984)), and by Barnesand Sato (Cell, 22:649 (1980)). Typically, the cells are capable ofexpressing and secreting large quantities of a particular protein ofinterest (typically a recombinant protein) into the culture medium, andare cultured for this purpose. However, the cells may be cultured for avariety of other purposes as well, and the scope of this invention isnot limited to culturing the cells only for production of recombinantproteins. Examples of suitable mammalian cell lines, capable of growthin the media of this invention, include monkey kidney CVI linetransformed by SV40 (COS-7, ATCC® CRL 1651); human embryonic kidney line293S (Graham et al., J. Gen. Virolo., 36:59 (1977)); baby hamster kidneycells (BHK, ATCC® CCL 10); mouse sertoli cells (TM4, Mather, Biol.Reprod., 23:243 (1980)); monkey kidney cells (CVI-76, ATCC® CCL 70);African green monkey kidney cells (VERO-76, ATCC® CRL-1587); humancervical carcinoma cells (HELA, ATCC® CCL 2); canine kidney cells (MDCK,ATCC® CCL 34); buffalo rat liver cells (BRL 3A, ATCC® CRL 1442); humanlung cells (W138, ATCC® CCL 75); human liver cells (Hep G2, HB 8065);mouse mammary tumor cells (MMT 060562, ATCC® CCL 5I); rat hepatoma cells(HTC, MI.54, Baumann et al., J. Cell Biol., 85:1 (1980)); and TR-1 cells(Mather et al., Annals N.Y. Acad. Sci., 383:44 (1982)) and hybridomacell lines. Chinese hamster ovary cells (Urlab and Chasin, Proc. Natl.Acad. Sci. USA, 77:4216 (1980)) are a preferred cell line for practicingthis invention. CHO cells suitable for use in the methods of the presentinvention have also been described in the following documents: EP117,159, published Aug. 29, 1989; U.S. Pat. Nos. 4,766,075; 4,853,330;5,185,259; Lubiniecki et al., in Advances in Animal Cell Biology andTechnology for Bioprocesses, Spier et al., eds. (1989), pp. 442-451.Known CHO derivatives suitable for use herein include, for example,CHO/-DHFR (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216(1980)), CHO-K1 DUX B11 (Simonsen and Levinson, Proc. Natl. Acad. Sci.USA 80: 2495-2499 (1983); Urlaub and Chasin, supra), and dp 12.CHO cells(EP 307,247 published Mar. 15, 1989). Preferred host cells includeCHO-K1 DUX B11 and dp 12.CHO cells.

“dhfr⁻ CHO cell” refers to a dihydrofolate reductase (DHFR) deficientCHO cell. Production of recombinant proteins in mammalian cells hasallowed the manufacture of a number of large, complex glycosylatedpolypeptides for clinical applications. Chinese hamster ovary (CHO)DHFR− cells and the amplifiable selectable marker DHFR are routinelyused to establish cell lines that produce clinically useful amounts ofproduct. (Urlab, G. and Chasin, L. A. (1980) Proc. Natl Acad. Sci. USA,77, 4216-4220; Kaufman, R. J. and Sharp, P. (1982) J. Mol. Biol., 159,601-621; Gasser, C. S., Simonsen, C. S., Schilling, J. W. and Schmike,R. T. (1982) Proc. Natl Sci. USA, 79, 6522-6526)

By “phase” is meant a certain phase of culturing of the cells as is wellrecognized by the practitioner.

“Growth phase” of the cell culture refers to the period of exponentialcell growth (the log phase) where cells are generally rapidly dividing.During this phase, cells are cultured for a period of time, usuallybetween 1-4 days, and under such conditions that cell growth ismaximized. The growth cycle for the host cell can be determined for theparticular host cell envisioned without undue experimentation. Duringthe growth phase, cells are cultured in nutrient medium containing thenecessary additives generally at about 30-40° C., preferably about 37°C., in a humidified, controlled atmosphere, such that optimal growth isachieved for the particular cell line. Cells are maintained in thegrowth phase for a period of between about one and four days, usuallybetween about two and three days.

“Transition phase” of the cell culture refers to the period of timeduring which culture conditions for the production phase are engaged.During the transition phase environmental factors such as temperatureare shifted from growth conditions to production conditions.

“Production phase” of the cell culture refers to the period of timeduring which cell growth has plateaued. During the production phase,logarithmic cell growth has ended and protein production is primary.During this period of time the medium is generally supplemented tosupport continued protein production and to achieve the desired proteinproduct.

The phrase “fed batch cell culture” when used herein refers to a batchculture wherein the animal (e.g. mammalian) cells and culture medium aresupplied to the culturing vessel initially and additional culturenutrients are fed, continuously or in discrete increments, to theculture during culturing, with or without periodic cell and/or productharvest before termination of culture. Fed batch culture includes“semi-continuous fed batch culture” wherein periodically whole culture(including cells and medium) is removed and replaced by fresh medium.Fed batch culture is distinguished from simple “batch culture” in whichall components for cell culturing (including the animal cells and allculture nutrients) are supplied to the culturing vessel at the start ofthe culturing process. Fed batch culture can be further distinguishedfrom perfusion culturing insofar as the supernatant is not removed fromthe culturing vessel during the process (in perfusion culturing, thecells are restrained in the culture by, e.g., filtration, encapsulation,anchoring to microcarriers etc and the culture medium is continuously orintermittently introduced and removed from the culturing vessel).However, removal of samples for testing purposes during fed batch cellculture is contemplated.

When used herein, the term “glutamine” refers to the amino acidL-glutamine (also known as “Gln” and “Q” by three-letter andsingle-letter designation, respectively) which is recognized as both anamino acid building block for protein synthesis and as an energy sourcein cell culture. Thus, the terms “glutamine” and “L-glutamine” are usedinterchangeably herein.

The word “glucose” refers to either of α-D-glucose or β-D-glucose,separately or in combination. It is noted that α and β glucose forms areinterconvertible in solution.

The expression “osmolality” is a measure of the osmotic pressure ofdissolved solute particles in an aqueous solution. The solute particlesinclude both ions and non-ionized molecules. Osmolality is expressed asthe concentration of osmotically active particles (i.e., osmoles)dissolved in 1 kg of water (1 mOsm/kg H₂O at 38° C. is equivalent to anosmotic pressure of 19 mm Hg). “Osmolarity” refers to the number ofsolute particles dissolved in 1 liter of solution. Solutes which can beadded to the culture medium so as to increase the osmolality thereofinclude proteins, peptides, amino acids, non-metabolized polymers,vitamins, ions, salts, sugars, metabolites, organic acids, lipids, etc.In the preferred embodiment, the concentration of amino acids and NaClin the culture medium is increased in order to achieve the desiredosmolality ranges set forth herein. When used herein, the abbreviation“mOsm” means “milliosmoles/kg H₂O”.

The term “cell density” as used herein refers to that number of cellspresent in a given volume of medium.

The term “cell viability” as used herein refers to the ability of cellsin culture to survive under a given set of culture conditions orexperimental variations. The term as used herein also refers to thatportion of cells which are alive at a particular time in relation to thetotal number of cells, living and dead, in the culture at that time.

The terms “amino acids” and “amino acid” refer to all naturallyoccurring alpha amino acids in both their D and L stereoisomeric forms,and their analogs and derivatives. An analog is defined as asubstitution of an atom in the amino acid with a different atom thatusually has similar properties. A derivative is defined as an amino acidthat has another molecule or atom attached to it. Derivatives wouldinclude, for example, acetylation of an amino group, amination of acarboxyl group, or oxidation of the sulfur residues of two cysteinemolecules to form cystine.

The term “protein” is meant to refer to a sequence of amino acids forwhich the chain length is sufficient to produce the higher levels oftertiary and/or quaternary structure. This is to distinguish from“peptides” or other small molecular weight drugs that do not have suchstructure. Typically, the protein herein will have a molecular weight ofat least about 15-20 kD, preferably at least about 20 kD. Examples ofproteins encompassed within the definition herein include all mammalianproteins, in particular, therapeutic and diagnostic proteins, such astherapeutic and diagnostic antibodies, and, in general proteins thatcontain one or more disulfide bonds, including multi-chain polypeptidescomprising one or more inter- and/or intrachain disulfide bonds.

The term “therapeutic protein” or “therapeutic polypeptide” refers to aprotein that is used in the treatment of disease, regardless of itsindication or mechanism of action. In order for therapeutic proteins tobe useful in the clinic it must be manufactured in large quantities.“Manufacturing scale” production of therapeutic proteins, or otherproteins, utilize cell cultures ranging from about 400 L to about 80,000L, depending on the protein being produced and the need. Typically suchmanufacturing scale production utilizes cell culture sizes from about400 L to about 25,000 L. Within this range, specific cell culture sizessuch as 4,000 L, about 6,000 L, about 8,000, about 10,000, about 12,000L, about 14,000 L, or about 16,000 L are utilized.

As used herein, “polypeptide of interest” refers generally to peptidesand proteins having more than about ten amino acids. The polypeptidesmay be homologous to the host cell, or preferably, may be exogenous,meaning that they are heterologous, i.e., foreign, to the host cellbeing utilized, such as a human protein produced by a non-humanmammalian, e.g., Chinese Hamster Ovary (CHO) cell. Preferably, mammalianpolypeptides (polypeptides that were originally derived from a mammalianorganism) are used, more preferably those which are directly secretedinto the medium. The term “polypeptide” or “polypeptide of interest”specifically includes antibodies, in particular, antibodies binding tomammalian polypeptides, such as any of the mammalian polypeptides listedbelow or fragments thereof, as well as immunoadhesins (polypeptide-Igfusion), such as those comprising any of the mammalian polypeptideslisted below, or fragments thereof.

Examples of mammalian polypeptides include, without limitation,transmembrane molecules (e.g. receptors) and ligands such, as growthfactors. Exemplary polypeptides include molecules such as renin; agrowth hormone, including human growth hormone and bovine growthhormone; growth hormone releasing factor; parathyroid hormone; thyroidstimulating hormone; interferon such as interferon-α, -β, and -γ;lipoproteins; α-1-antitrypsin; insulin A-chain; insulin B-chain;proinsulin; follicle stimulating hormone; calcitonin; luteinizinghormone; glucagon; clotting factors such as factor VIIIC, factor IX,tissue factor, and von Willebrands factor; anti-clotting factors such asProtein C; atrial natriuretic factor; lung surfactant; a plasminogenactivator, such as urokinase or human urine or tissue-type plasminogenactivator (t-PA), including t-PA variants; bombesin; thrombin;hemopoietic growth factor; tumor necrosis factor-alpha and -beta;enkephalinase; RANTES (regulated on activation normally T-cell expressedand secreted); human macrophage inflammatory protein (MIP-1-α); a serumalbumin such as human serum albumin; Muellerian-inhibiting substance;relaxin A-chain; relaxin B-chain; prorelaxin; mousegonadotropin-associated peptide; a microbial protein, such asβ-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associated antigen(CTLA), such as CTLA-4; inhibin; activin; vascular endothelial growthfactor (VEGF); receptors for hormones or growth factors; protein A or D;rheumatoid factors; a neurotrophic factor such as bone-derivedneurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT4,NT-5, or NT-6), or a nerve growth factor such as NGF-β; platelet-derivedgrowth factor (PDGF); fibroblast growth factor such as aFGF and bFGF;epidermal growth factor (EGF); transforming growth factor (TGF) such asTGF-α and TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5;insulin-like growth factor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I(brain IGF-I), insulin-like growth factor binding proteins; CD proteinssuch as CD3, CD4, CD8, CD19, CD20, CD34, CD40; erythropoietin;osteoinductive factors; immunotoxins; a bone morphogenetic protein(BMP); an interferon such as interferon-α, -β, and -γ; colonystimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins(ILs), e.g., IL-1 to IL-10; superoxide dismutase; T-cell receptors;surface membrane proteins; decay accelerating factor; viral antigen suchas, for example, a portion of the AIDS envelope; transport proteins;homing receptors; addressins; regulatory proteins; integrins such asCD11a, CD11b, CD11c, CD18, an ICAM, VLA-4 and VCAM; a tumor associatedantigen such as HER1 (EGFR), HER2, HER3 or HER4 receptor; Apo2L/TRAIL,hedgehog, mitogen activated protein kinase (MAPK), and fragments of anyof the above-listed polypeptides. Apo2L (TRAIL) and is variants aredisclosed, for example, in U.S. Application Publication No. 20040186051.Anti-VEGF antibodies are disclosed, for example, in U.S. Pat. Nos.8,994,879; 7,060,269; 7,169,901; and 7,297,334. Anti-CD20 antibodies aredisclosed, for example, in U.S. Application Publication No. 20060246004.The BR3 polypeptide, anti-BR3 antibodies and BR3-Fc immunoadhesins aredescribed, for example, in U.S. Application Publication No. 20050070689.

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

As noted above, in certain embodiments, the protein is an antibody.“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which generally lackantigen specificity. Polypeptides of the latter kind are, for example,produced at low levels by the lymph system and at increased levels bymyelomas.

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length antibodies whichhave an immunoglobulin Fc region or intact monoclonal antibodies),antibody compositions with polyepitopic specificity, polyclonalantibodies, multivalent antibodies, multispecific antibodies (e.g.,bispecific antibodies) formed from at least two intact antibodies,diabodies, and single-chain molecules such as scFv molecules, as well asantibody fragments (e.g., Fab, F(ab′)2, and Fv).

Unless indicated otherwise, the expression “multivalent antibody” isused throughout this specification to denote an antibody comprisingthree or more antigen binding sites. The multivalent antibody istypically engineered to have the three or more antigen binding sites andis generally not a native sequence IgM or IgA antibody.

The terms “full length antibody,” “intact antibody” and “whole antibody”are used herein interchangeably to refer to an antibody in itssubstantially intact form, not antibody fragments as defined below. Theterms particularly refer to an antibody with heavy chains that containthe Fc region.

“Antibody fragments” comprise only a portion of an intact antibody,generally including an antigen binding site of the intact antibody andthus retaining the ability to bind antigen. In one embodiment, anantibody fragment comprises an antigen binding site of the intactantibody and thus retains the ability to bind antigen. In anotherembodiment, an antibody fragment, for example one that comprises the Fcregion, retains at least one of the biological functions normallyassociated with the Fc region when present in an intact antibody, suchas FcRn binding, antibody half life modulation, ADCC function andcomplement binding. In one embodiment, an antibody fragment is amonovalent antibody that has an in vivo half life substantially similarto an intact antibody. For example, such an antibody fragment maycomprise an antigen binding arm linked to an Fc sequence capable ofconferring in vivo stability to the fragment.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fe” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)2 fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

The Fab fragment contains the heavy- and light-chain variable domainsand also contains the constant domain of the light chain and the firstconstant domain (CH1) of the heavy chain. Fab′ fragments differ from Fabfragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CH1 domain including one or more cysteines from theantibody hinge region. Fab′-SH is the designation herein for Fab′ inwhich the cysteine residue(s) of the constant domains bear a free thiolgroup. F(ab′)2 antibody fragments originally were produced as pairs ofFab′ fragments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known. Examples of antibodyfragments encompassed by the present definition include: (i) the Fabfragment, having VL, CL, VH and CH1 domains; (ii) the Fab′ fragment,which is a Fab fragment having one or more cysteine residues at theC-terminus of the CH1 domain; (iii) the Fd fragment having VH and CH1domains; (iv) the Fd′ fragment having VH and CH1 domains and one or morecysteine residues at the C-terminus of the CH1 domain; (v) the Fvfragment having the VL and VH domains of a single arm of an antibody;(vi) the dAb fragment (Ward et al., Nature 341, 544-546 (1989)) whichconsists of a VH domain; (vii) isolated CDR regions; (viii) F(ab′)2fragments, a bivalent fragment including two Fab′ fragments linked by adisulphide bridge at the hinge region; (ix) single chain antibodymolecules (e.g. single chain Fv; scFv) (Bird et al., Science 242:423-426(1988); and Huston et al., PNAS (USA) 85:5879-5883 (1988)); (x)“diabodies” with two antigen binding sites, comprising a heavy chainvariable domain (VH) connected to a light chain variable domain (VL) inthe same polypeptide chain (see, e.g., EP 404,097; WO 93/11161; andHollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); (xi)“linear antibodies” comprising a pair of tandem Fd segments(VH-CH1-VH-CH1) which, together with complementary light chainpolypeptides, form a pair of antigen binding regions (Zapata et al.Protein Eng. 8(10):1057 1062 (1995); and U.S. Pat. No. 5,641,870).

“Fv” is the minimum antibody fragment which contains a completeantigen-binding site. In one embodiment, a two-chain Fv species consistsof a dimer of one heavy- and one light-chain variable domain in tight,non-covalent association. In a single-chain Fv (scFv) species, oneheavy- and one light-chain variable domain can be covalently linked by aflexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of an antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. For a review of scFvsee Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies may be bivalent orbispecific. Diabodies are described more fully in, for example, EP404,097; W093/1161; Hudson et al., (2003) Nat. Med. 9:129-134; andHollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).Triabodies and tetrabodies are also described in Hudson et al., (2003)Nat. Med. 9:129-134.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible mutations, e.g., naturally occurring mutations, thatmay be present in minor amounts. Thus, the modifier “monoclonal”indicates the character of the antibody as not being a mixture ofdiscrete antibodies. Monoclonal antibodies are highly specific, beingdirected against a single antigen. In certain embodiments, a monoclonalantibody typically includes an antibody comprising a polypeptidesequence that binds a target, wherein the target-binding polypeptidesequence was obtained by a process that includes the selection of asingle target binding polypeptide sequence from a plurality ofpolypeptide sequences. For example, the selection process can be theselection of a unique clone from a plurality of clones, such as a poolof hybridoma clones, phage clones, or recombinant DNA clones. It shouldbe understood that a selected target binding sequence can be furtheraltered, for example, to improve affinity for the target, to humanizethe target binding sequence, to improve its production in cell culture,to reduce its immunogenicity in vivo, to create a multispecificantibody, etc., and that an antibody comprising the altered targetbinding sequence is also a monoclonal antibody of this invention. Incontrast to polyclonal antibody preparations that typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, monoclonal antibodypreparations are advantageous in that they are typically uncontaminatedby other immunoglobulins.

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by a variety of techniques, including, for example, the hybridomamethod (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo etal., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567), phage-display technologies (see, e.g.,Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol.Biol. 222: 581-597 (1991); Sidhu et al., J. Mol. Biol. 338(2): 299-310(2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse,Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al.,J. Immunol. Methods 284(1-2): 119-132 (2004), and technologies forproducing human or human-like antibodies in animals that have parts orall of the human immunoglobulin loci or genes encoding humanimmunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993);Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016;Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild etal., Nature Biotechnol. 14: 845-851 (1996); Neuberger, NatureBiotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev.Immunol. 13: 65-93 (1995).

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, e.g.,Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998);Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross,Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and7,087,409. See also van Dijk and van de Winkel, Curr. Opin. Pharmacol.,5: 368-74 (2001). Human antibodies can be prepared by administering theantigen to a transgenic animal that has been modified to produce suchantibodies in response to antigenic challenge, but whose endogenous locihave been disabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos.6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). See also, forexample, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006)regarding human antibodies generated via a human B-cell hybridomatechnology. The humanized antibody may also include a Primatized™antibody wherein the antigen-binding region of the antibody is derivedfrom an antibody produced by immunizing macaque monkeys with the antigenof interest.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues. Human antibodies can be produced using various techniquesknown in the art. In one embodiment, the human antibody is selected froma phage library, where that phage library expresses human antibodies(Vaughan et al. Nature Biotechnology 14:309-314 (1996): Sheets et al.PNAS (USA) 95:6157-6162 (1998)); Hoogenboom and Winter, J. Mol. Biol.,227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). Humanantibodies can also be made by introducing human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10: 779-783(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature368:812-13 (1994); Fishwild et al., Nature Biotechnology 14: 845-51(1996); Neuberger, Nature Biotechnology 14: 826 (1996); Lonberg andHuszar, Intern. Rev. Immunol. 13:65-93 (1995). Alternatively, the humanantibody may be prepared via immortalization of human B lymphocytesproducing an antibody directed against a target antigen (such Blymphocytes may be recovered from an individual or may have beenimmunized in vitro). See, e.g., Cole et al., Monoclonal Antibodies andCancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol.,147 (1):86-95 (1991); and U.S. Pat. No. 5,750,373.

An “affinity matured” antibody is one with one or more alterations inone or more CDRs/HVRs thereof which result in an improvement in theaffinity of the antibody for antigen, compared to a parent antibodywhich does not possess those alteration(s). Preferred affinity maturedantibodies will have nanomolar or even picomolar affinities for thetarget antigen. Affinity matured antibodies are produced by proceduresknown in the art. Marks et al., Bio/Technology 10:779-783 (1992)describes affinity maturation by VH and VL domain shuffling. Randommutagenesis of CDR/HVR and/or framework residues is described by: Barbaset al., Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al.,Gene 169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004(1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins etal., J. Mol. Biol. 226:889-896 (1992).

The “variable region” or “variable domain” of an antibody refers to theamino-terminal domains of the heavy or light chain of the antibody. Thevariable domain of the heavy chain may be referred to as “VH.” Thevariable domain of the light chain may be referred to as “VL.” Thesedomains are generally the most variable parts of an antibody and containthe antigen-binding sites.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions (HVRs) both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework regions (FRs). The variable domains of nativeheavy and light chains each comprise four FRs, largely adopting abeta-sheet configuration, connected by three hypervariable regions,which form loops connecting, and in some cases forming part of, thebeta-sheet structure. The hypervariable regions in each chain are heldtogether in close proximity by the FRs and, with the hypervariableregions from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). The constantdomains are not involved directly in binding an antibody to an antigen,but exhibit various effector functions, such as participation of theantibody in antibody-dependent cellular toxicity.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the amino acid residues of an antibody which are responsible forantigen-binding. For example, the term hypervariable region refers tothe regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six HVRs; three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). In native antibodies, H3 and L3 display the most diversityof the six HVRs, and H3 in particular is believed to play a unique rolein conferring fine specificity to antibodies. See, e.g., Xu et al.,Immunity 13:37-45 (2000); Johnson and Wu, in Methods in MolecularBiology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed,naturally occurring camelid antibodies consisting of a heavy chain onlyare functional and stable in the absence of light chain. See, e.g.,Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al.,Nature Struct. Biol. 3:733-736 (1996).

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequences of the constant domains of theirheavy chains, antibodies (immunoglobulins) can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG and IgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavychain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known and described generally in, for example,Abbas et al., Cellular and Mol. Immunology, 4th ed. (2000). An antibodymay be part of a larger fusion molecule, formed by covalent ornon-covalent association of the antibody with one or more other proteinsor peptides.

The term “Fc region” is used to define the C-terminal region of animmunoglobulin heavy chain which may be generated by papain digestion ofan intact antibody. The Fc region may be a native sequence Fc region ora variant Fc region. The Fc region of an immunoglobulin generallycomprises two constant domains, a CH2 domain and a CH3 domain, andoptionally comprises a CH4 domain.

By “Fc region chain” herein is meant one of the two polypeptide chainsof an Fc region.

The “CH2 domain” of a human IgG Fc region (also referred to as “Cg2”domain) is unique in that it is not closely paired with another domain.Rather, two N-linked branched carbohydrate chains are interposed betweenthe two CH2 domains of an intact native IgG molecule. It has beenspeculated that the carbohydrate may provide a substitute for thedomain-domain pairing and help stabilize the CH2 domain. Burton, Molec.Immunol. 22:161-206 (1985). The CH2 domain herein may be a nativesequence CH2 domain or variant CH2 domain.

The “CH3 domain” comprises the stretch of residues C-terminal to a CH2domain in an Fc region. The CH3 region herein may be a native sequenceCH3 domain or a variant CH3 domain (e.g. a CH3 domain with an introduced“protroberance” in one chain thereof and a corresponding introduced“cavity” in the other chain thereof; see U.S. Pat. No. 5,821,333,expressly incorporated herein by reference). Such variant CH3 domainsmay be used to make multispecific (e.g. bispecific) antibodies as hereindescribed.

“Hinge region” herein may be a native sequence hinge region or a varianthinge region. The two polypeptide chains of a variant hinge regiongenerally retain at least one cysteine residue per polypeptide chain, sothat the two polypeptide chains of the variant hinge region can form adisulfide bond between the two chains. The preferred hinge region hereinis a native sequence human hinge region, e.g. a native sequence humanIgG1 hinge region.

A “functional Fc region” possesses at least one “effector function” of anative sequence Fc region. Exemplary “effector functions” include C1qbinding; complement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor; BCR), etc.Such effector functions generally require the Fc region to be combinedwith a binding domain (e.g. an antibody variable domain) and can beassessed using various assays known in the art for evaluating suchantibody effector functions.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. Nativesequence human Fc regions include a native sequence human IgG1 Fc region(non-A and A allotypes); native sequence human IgG2 Fc region; nativesequence human IgG3 Fc region; and native sequence human IgG4 Fc regionas well as naturally occurring variants thereof.

An “intact” antibody is one which comprises an antigen-binding variableregion as well as a light chain constant domain (C_(L)) and heavy chainconstant domains, C_(H)1, C_(H)2 and C_(H)3. The constant domains may benative sequence constant domains (e.g. human native sequence constantdomains) or amino acid sequence variant thereof. Preferably, the intactantibody has one or more effector functions.

A “parent antibody” or “wild-type” antibody is an antibody comprising anamino acid sequence which lacks one or more amino acid sequencealterations compared to an antibody variant as herein disclosed. Thus,the parent antibody generally has at least one hypervariable regionwhich differs in amino acid sequence from the amino acid sequence of thecorresponding hypervariable region of an antibody variant as hereindisclosed. The parent polypeptide may comprise a native sequence (i.e. anaturally occurring) antibody (including a naturally occurring allelicvariant), or an antibody with pre-existing amino acid sequencemodifications (such as insertions, deletions and/or other alterations)of a naturally occurring sequence. Throughout the disclosure, “wildtype,” “WT,” “wt,” and “parent” or “parental” antibody are usedinterchangeably.

As used herein, “antibody variant” or “variant antibody” refers to anantibody which has an amino acid sequence which differs from the aminoacid sequence of a parent antibody. Preferably, the antibody variantcomprises a heavy chain variable domain or a light chain variable domainhaving an amino acid sequence which is not found in nature. Suchvariants necessarily have less than 100% sequence identity or similaritywith the parent antibody. In a preferred embodiment, the antibodyvariant will have an amino acid sequence from about 75% to less than100% amino acid sequence identity or similarity with the amino acidsequence of either the heavy or light chain variable domain of theparent antibody, more preferably from about 80% to less than 100%, morepreferably from about 85% to less than 100%, more preferably from about90% to less than 100%, and most preferably from about 95% to less than100%. The antibody variant is generally one which comprises one or moreamino acid alterations in or adjacent to one or more hypervariableregions thereof.

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a native sequence Fc region by virtue of at least one aminoacid modification. In certain embodiments, the variant Fc region has atleast one amino acid substitution compared to a native sequence Fcregion or to the Fc region of a parent polypeptide, e.g. from about oneto about ten amino acid substitutions, and preferably from about one toabout five amino acid substitutions in a native sequence Fc region or inthe Fc region of the parent polypeptide, e.g. from about one to aboutten amino acid substitutions, and preferably from about one to aboutfive amino acid substitutions in a native sequence Fc region or in theFc region of the parent polypeptide. The variant Fc region herein willtypically possess, e.g., at least about 80% sequence identity with anative sequence Fc region and/or with an Fc region of a parentpolypeptide, or at least about 90% sequence identity therewith, or atleast about 95% sequence or more identity therewith.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g. Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The primary cells for mediatingADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI,FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarizedin Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92(1991). To assess ADCC activity of a molecule of interest, an in vitroADCC assay, such as that described in U.S. Pat. No. 5,500,362 or U.S.Pat. No. 5,821,337 may be performed. Useful effector cells for suchassays include peripheral blood mononuclear cells (PBMC) and NaturalKiller (NK) cells. Alternatively, or additionally, ADCC activity of themolecule of interest may be assessed in vivo, e.g., in a animal modelsuch as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. In certain embodiments, the cells express atleast FcγRIII and perform ADCC effector function(s). Examples of humanleukocytes which mediate ADCC include peripheral blood mononuclear cells(PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells andneutrophils; with PBMCs and NK cells being generally preferred. Theeffector cells may be isolated from a native source thereof, e.g. fromblood or PBMCs as described herein.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. In some embodiments, an FcR is a native human FcR. Insome embodiments, an FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof those receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domainInhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see, e.g., Daëron,Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example,in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al.,Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126:330-41 (1995). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein.

The term “Fc receptor” or “FcR” also includes the neonatal receptor,FcRn, which is responsible for the transfer of maternal IgGs to thefetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J.Immunol. 24:249 (1994)) and regulation of homeostasis ofimmunoglobulins. Methods of measuring binding to FcRn are known (see,e.g., Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie etal., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol.Chem. 279(8):6213-6216 (2004); WO 2004/92219 (Hinton et al.).

Binding to human FcRn in vivo and serum half life of human FcRn highaffinity binding polypeptides can be assayed, e.g., in transgenic miceor transfected human cell lines expressing human FcRn, or in primates towhich the polypeptides with a variant Fc region are administered. WO2000/42072 (Presta) describes antibody variants with improved ordiminished binding to FcRs. See also, e.g., Shields et al. J. Biol.Chem. 9(2):6591-6604 (2001).

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (Clq) to antibodies (of the appropriate subclass),which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g., as described in Gazzano-Santoro et al.,J. Immunol. Methods 202:163 (1996), may be performed. Polypeptidevariants with altered Fc region amino acid sequences (polypeptides witha variant Fc region) and increased or decreased C1q binding capabilityare described, e.g., in U.S. Pat. No. 6,194,551 B1 and WO 1999/51642.See also, e.g., Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

An “affinity matured” antibody is one with one or more alterations inone or more CDRs thereof which result an improvement in the affinity ofthe antibody for antigen, compared to a parent antibody which does notpossess those alteration(s). In one embodiment, an affinity maturedantibody has nanomolar or even picomolar affinities for the targetantigen. Affinity matured antibodies are produced by procedures known inthe art. Marks et al. Bio/Technology 10:779-783 (1992) describesaffinity maturation by VH and VL domain shuffling. Random mutagenesis ofCDR and/or framework residues is described by: Barbas et al. Proc Nat.Acad. Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155(1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al.,J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol.226:889-896 (1992).

The term “therapeutic antibody” refers to an antibody that is used inthe treatment of disease. A therapeutic antibody may have variousmechanisms of action. A therapeutic antibody may bind and neutralize thenormal function of a target associated with an antigen. For example, amonoclonal antibody that blocks the activity of the of protein neededfor the survival of a cancer cell causes the cell's death. Anothertherapeutic monoclonal antibody may bind and activate the normalfunction of a target associated with an antigen. For example, amonoclonal antibody can bind to a protein on a cell and trigger anapoptosis signal. Yet another monoclonal antibody may bind to a targetantigen expressed only on diseased tissue; conjugation of a toxicpayload (effective agent), such as a chemotherapeutic or radioactiveagent, to the monoclonal antibody can create an agent for specificdelivery of the toxic payload to the diseased tissue, reducing harm tohealthy tissue. A “biologically functional fragment” of a therapeuticantibody will exhibit at least one if not some or all of the biologicalfunctions attributed to the intact antibody, the function comprising atleast specific binding to the target antigen.

The antibody may bind to any protein, including, without limitation, amember of the HER receptor family, such as HER1 (EGFR), HER2, HER3 andHER4; CD proteins such as CD3, CD4, CD8, CD19, CD20, CD21, CD22, andCD34; cell adhesion molecules such as LFA-1, Mol, p150,95, VLA-4,ICAM-1, VCAM and av/p3 integrin including either α or β or subunitsthereof (e.g. anti-CD11a, anti-CD18 or anti-CD11b antibodies); growthfactors such as vascular endothelial growth factor (VEGF); IgE; bloodgroup antigens; flk2/flt3 receptor; obesity (OB) receptor; and proteinC. Other exemplary proteins include growth hormone (GH), including humangrowth hormone (hGH) and bovine growth hormone (bGH); growth hormonereleasing factor; parathyroid hormone; thyroid stimulating hormone;lipoproteins; α-1-antitrypsin; insulin A-chain; insulin B-chain;proinsulin; follicle stimulating hormone; calcitonin; luteinizinghormone; glucagon; clotting factors such as factor VIIIC, factor, tissuefactor, and von Willebrands factor; anti-clotting factors such asProtein C; atrial natriuretic factor; lung surfactant; a plasminogenactivator, such as urokinase or tissue-type plasminogen activator(t-PA); bombazine; thrombin; tumor necrosis factor-α and -β;enkephalinase; RANTES (regulated on activation normally T-cell expressedand secreted); human macrophage inflammatory protein (MIP-1-α); serumalbumin such as human serum albumin (HSA); mullerian-inhibitingsubstance; relaxin A-chain; relaxin B-chain; prorelaxin; mousegonadotropin-associated peptide; DNase; inhibin; activin; receptors forhormones or growth factors; an integrin; protein A or D; rheumatoidfactors; a neurotrophic factor such as bone-derived neurotrophic factor(BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or anerve growth factor such as NGF-β; platelet-derived growth factor(PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growthfactor (EGF); transforming growth factor (TGF) such as TGF-α and TGF-β,including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growthfactor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I);insulin-like growth factor binding proteins (IGFBPs); erythropoietin(EPO); thrombopoietin (TPO); osteoinductive factors; immunotoxins; abone morphogenetic protein (BMP); an interferon such as interferon-α,-β, and -γ; colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, andG-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxide dismutase;T-cell receptors; surface membrane proteins; decay accelerating factor(DAF); a viral antigen such as, for example, a portion of the AIDSenvelope; transport proteins; homing receptors; addressins; regulatoryproteins; immunoadhesins; antibodies; and biologically active fragmentsor variants of any of the above-listed polypeptides. Many otherantibodies and/or other proteins may be used in accordance with theinstant invention, and the above lists are not meant to be limiting.

Therapeutic antibodies of particular interest include those in clinicaloncological practice or development such as commercially availableAVASTIN® (bevacizumab), HERCEPTIN® (trastuzumab), LUCENTIS®(ranibizumab), RAPTIVA® (efalizumab), RITUXAN® (rituximab), and XOLAIR®(omalizumab), as well as, anti-amyloid beta (Abeta), anti-CD4(MTRX1011A), anti-EGFL7 (EGF-like-domain 7), anti-IL13, Apomab(anti-DR5-targeted pro-apoptotic receptor agonist (PARA), anti-BR3(CD268, BLyS receptor 3, BAFF-R, BAFF Receptor), anti-beta 7 integrinsubunit, dacetuzumab (Anti-CD40), GA101 (anti-CD20 monoclonal antibody),MetMAb (anti-MET receptor tyrosine kinase), anti-neuropilin-1 (NRP1),ocrelizumab (anti-CD20 antibody), anti-OX40 ligand, anti-oxidized LDL(oxLDL), pertuzumab (HER dimerization inhibitors (HDIs), and. rhuMAb IFNalpha.

A “biologically functional fragment” of an antibody comprises only aportion of an intact antibody, wherein the portion retains at least one,and as many as most or all, of the functions normally associated withthat portion when present in an intact antibody. In one embodiment, abiologically functional fragment of an antibody comprises an antigenbinding site of the intact antibody and thus retains the ability to bindantigen. In another embodiment, a biologically functional fragment of anantibody, for example one that comprises the Fc region, retains at leastone of the biological functions normally associated with the Fc regionwhen present in an intact antibody, such as FcRn binding, antibody halflife modulation, ADCC function and complement binding. In oneembodiment, a biologically functional fragment of an antibody is amonovalent antibody that has an in vivo half life substantially similarto an intact antibody. For example, such a biologically functionalfragment of an antibody may comprise an antigen binding arm linked to anFc sequence capable of conferring in vivo stability to the fragment.

The term “diagnostic protein” refers to a protein that is used in thediagnosis of a disease.

The term “diagnostic antibody” refers to an antibody that is used as adiagnostic reagent for a disease. The diagnostic antibody may bind to atarget antigen that is specifically associated with, or shows increasedexpression in, a particular disease. The diagnostic antibody may beused, for example, to detect a target in a biological sample from apatient, or in diagnostic imaging of disease sites, such as tumors, in apatient. A “biologically functional fragment” of a diagnostic antibodywill exhibit at least one if not some or all of the biological functionsattributed to the intact antibody, the function comprising at leastspecific binding to the target antigen.

“Purified” means that a molecule is present in a sample at aconcentration of at least 80-90% by weight of the sample in which it iscontained. The protein, including antibodies, which is purified ispreferably essentially pure and desirably essentially homogeneous (i.e.free from contaminating proteins etc.).

An “essentially pure” protein means a protein composition comprising atleast about 90% by weight of the protein, based on total weight of thecomposition, preferably at least about 95% by weight.

An “essentially homogeneous” protein means a protein compositioncomprising at least about 99% by weight of protein, based on totalweight of the composition.

As used herein, “soluble” refers to polypeptides that, when in aqueoussolutions, are completely dissolved, resulting in a clear to slightlyopalescent solution with no visible particulates, as assessed by visualinspection. A further assay of the turbidity of the solution (orsolubility of the protein) may be made by measuring UV absorbances at340 nm to 360 nm with a 1 cm path-length cell where turbidity at 20mg/ml is less than 0.05 absorbance units.

An “isolated” antibody or polypeptide is one which has been identifiedand separated and/or recovered from a component of its naturalenvironment. Contaminant components of its natural environment arematerials which would interfere with research, diagnostic or therapeuticuses for the antibody, and may include enzymes, hormones, and otherproteinaceous or nonproteinaceous solutes. In some embodiments, anantibody is purified (1) to greater than 95% by weight of antibody asdetermined by, for example, the Lowry method, and in some embodiments,to greater than 99% by weight; (2) to a degree sufficient to obtain atleast 15 residues of N-terminal or internal amino acid sequence by useof, for example, a spinning cup sequenator, or (3) to homogeneity bySDS-PAGE under reducing or nonreducing conditions using, for example,Coomassie blue or silver stain. Isolated antibody includes the antibodyin situ within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step.

The terms “Protein A” and “ProA” are used interchangeably herein andencompasses Protein A recovered from a native source thereof, Protein Aproduced synthetically (e.g. by peptide synthesis or by recombinanttechniques), and variants thereof which retain the ability to bindproteins which have a C_(H)2/C_(H)3 region, such as an Fc region.Protein A can be purchased commercially from Repligen, Pharmacia andFermatech. Protein A is generally immobilized on a solid phase supportmaterial. The term “ProA” also refers to an affinity chromatographyresin or column containing chromatographic solid support matrix to whichis covalently attached Protein A.

The term “chromatography” refers to the process by which a solute ofinterest in a mixture is separated from other solutes in a mixture as aresult of differences in rates at which the individual solutes of themixture migrate through a stationary medium under the influence of amoving phase, or in bind and elute processes.

The term “affinity chromatography” and “protein affinity chromatography”are used interchangeably herein and refer to a protein separationtechnique in which a protein of interest or antibody of interest isreversibly and specifically bound to a biospecific ligand. Preferably,the biospecific ligand is covalently attached to a chromatographic solidphase material and is accessible to the protein of interest in solutionas the solution contacts the chromatographic solid phase material. Theprotein of interest (e.g., antibody, enzyme, or receptor protein)retains its specific binding affinity for the biospecific ligand(antigen, substrate, cofactor, or hormone, for example) during thechromatographic steps, while other solutes and/or proteins in themixture do not bind appreciably or specifically to the ligand. Bindingof the protein of interest to the immobilized ligand allowscontaminating proteins or protein impurities to be passed through thechromatographic medium while the protein of interest remainsspecifically bound to the immobilized ligand on the solid phasematerial. The specifically bound protein of interest is then removed inactive form from the immobilized ligand with low pH, high pH, high salt,competing ligand, and the like, and passed through the chromatographiccolumn with the elution buffer, free of the contaminating proteins orprotein impurities that were earlier allowed to pass through the column.Any component can be used as a ligand for purifying its respectivespecific binding protein, e.g. antibody.

The terms “non-affinity chromatography” and “non-affinity purification”refer to a purification process in which affinity chromatography is notutilized. Non-affinity chromatography includes chromatographictechniques that rely on non-specific interactions between a molecule ofinterest (such as a protein, e.g. antibody) and a solid phase matrix.

A “cation exchange resin” refers to a solid phase which is negativelycharged, and which thus has free cations for exchange with cations in anaqueous solution passed over or through the solid phase. A negativelycharged ligand attached to the solid phase to form the cation exchangeresin may, e.g., be a carboxylate or sulfonate. Commercially availablecation exchange resins include carboxy-methyl-cellulose, sulphopropyl(SP) immobilized on agarose (e.g. SP-SEPHAROSE FAST FLOW™ orSP-SEPHAROSE HIGH PERFORMANCE™, from Pharmacia) and sulphonylimmobilized on agarose (e.g. S-SEPHAROSE FAST FLOW™ from Pharmacia). A“mixed mode ion exchange resin” refers to a solid phase which iscovalently modified with cationic, anionic, and hydrophobic moieties. Acommercially available mixed mode ion exchange resin is BAKERBOND ABX™(J.T. Baker, Phillipsburg, N.J.) containing weak cation exchange groups,a low concentration of anion exchange groups, and hydrophobic ligandsattached to a silica gel solid phase support matrix.

The term “anion exchange resin” is used herein to refer to a solid phasewhich is positively charged, e.g. having one or more positively chargedligands, such as quaternary amino groups, attached thereto. Commerciallyavailable anion exchange resins include DEAE cellulose, QAE SEPHADEX™and FAST Q SEPHAROSE™ (Pharmacia).

A “buffer” is a solution that resists changes in pH by the action of itsacid-base conjugate components. Various buffers which can be employeddepending, for example, on the desired pH of the buffer are described inBuffers. A Guide for the Preparation and Use of Buffers in BiologicalSystems, Gueffroy, D., ed. Calbiochem Corporation (1975). In oneembodiment, the buffer has a pH in the range from about 2 to about 9,alternatively from about 3 to about 8, alternatively from about 4 toabout 7 alternatively from about 5 to about 7. Non-limiting examples ofbuffers that will control the pH in this range include MES, MOPS, MOPSO,Tris, HEPES, phosphate, acetate, citrate, succinate, and ammoniumbuffers, as well as combinations of these.

The “loading buffer” is that which is used to load the compositioncomprising the polypeptide molecule of interest and one or moreimpurities onto the ion exchange resin. The loading buffer has aconductivity and/or pH such that the polypeptide molecule of interest(and generally one or more impurities) is/are bound to the ion exchangeresin or such that the protein of interest flows through the columnwhile the impurities bind to the resin.

The “intermediate buffer” is used to elute one or more impurities fromthe ion exchange resin, prior to eluting the polypeptide molecule ofinterest. The conductivity and/or pH of the intermediate buffer is/aresuch that one or more impurity is eluted from the ion exchange resin,but not significant amounts of the polypeptide of interest.

The term “wash buffer” when used herein refers to a buffer used to washor re-equilibrate the ion exchange resin, prior to eluting thepolypeptide molecule of interest. Conveniently, the wash buffer andloading buffer may be the same, but this is not required.

The “elution buffer” is used to elute the polypeptide of interest fromthe solid phase. The conductivity and/or pH of the elution buffer is/aresuch that the polypeptide of interest is eluted from the ion exchangeresin.

A “regeneration buffer” may be used to regenerate the ion exchange resinsuch that it can be re-used. The regeneration buffer has a conductivityand/or pH as required to remove substantially all impurities and thepolypeptide of interest from the ion exchange resin.

The term “substantially similar” or “substantially the same,” as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (for example, one associated with an antibody of theinvention and the other associated with a reference/comparatorantibody), such that one of skill in the art would consider thedifference between the two values to be of little or no biologicaland/or statistical significance within the context of the biologicalcharacteristic measured by said values (e.g., Kd values). The differencebetween said two values is, for example, less than about 50%, less thanabout 40%, less than about 30%, less than about 20%, and/or less thanabout 10% as a function of the reference/comparator value.

The phrase “substantially reduced,” or “substantially different,” asused herein with regard to amounts or numerical values (and not asreference to the chemical process of reduction), denotes a sufficientlyhigh degree of difference between two numeric values (generally oneassociated with a molecule and the other associated with areference/comparator molecule) such that one of skill in the art wouldconsider the difference between the two values to be of statisticalsignificance within the context of the biological characteristicmeasured by said values (e.g., Kd values). The difference between saidtwo values is, for example, greater than about 10%, greater than about20%, greater than about 30%, greater than about 40%, and/or greater thanabout 50% as a function of the value for the reference/comparatormolecule.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA into which additional DNA segments may beligated. Another type of vector is a phage vector. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors,” or simply, “expressionvectors.” In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, preferably digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Y

-   -   where X is the number of amino acid residues scored as identical        matches by the sequence alignment program ALIGN-2 in that        program's alignment of A and B, and    -   where Y is the total number of amino acid residues in B.

It will be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

“Percent (%) nucleic acid sequence identity” is defined as thepercentage of nucleotides in a candidate sequence that are identicalwith the nucleotides in a reference Factor D-encoding sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity. Alignment for purposes ofdetermining percent nucleic acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared. Sequence identity is then calculated relativeto the longer sequence, i.e. even if a shorter sequence shows 100%sequence identity with a portion of a longer sequence, the overallsequence identity will be less than 100%.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented. “Treatment” herein encompasses alleviation of the disease andof the signs and symptoms of the particular disease.

A “disorder” is any condition that would benefit from treatment with theprotein. This includes chronic and acute disorders or diseases includingthose pathological conditions which predispose the mammal to thedisorder in question. Non-limiting examples of disorders to be treatedherein include carcinomas and allergies.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, non-human higher primates, other vertebrates,domestic and farm animals, and zoo, sports, or pet animals, such asdogs, horses, cats, cows, etc. Preferably, the mammal is human.

B. Exemplary Methods and Materials for Carrying Out the Invention

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology and the like,which are within the skill of the art. Such techniques are explainedfully in the literature. See e.g., Molecular Cloning: A LaboratoryManual, (J. Sambrook et al., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989); Current Protocols in Molecular Biology (F. Ausubelet al., eds., 1987 updated); Essential Molecular Biology (T. Brown ed.,IRL Press 1991); Gene Expression Technology (Goeddel ed., Academic Press1991); Methods for Cloning and Analysis of Eukaryotic Genes (A. Bothwellet al., eds., Bartlett Publ. 1990); Gene Transfer and Expression (M.Kriegler, Stockton Press 1990); Recombinant DNA Methodology II (R. Wu etal., eds., Academic Press 1995); PCR: A Practical Approach (M. McPhersonet al., IRL Press at Oxford University Press 1991); OligonucleotideSynthesis (M. Gait ed., 1984); Cell Culture for Biochemists (R. Adamsed., Elsevier Science Publishers 1990); Gene Transfer Vectors forMammalian Cells (J. Miller & M. Calos eds., 1987); Mammalian CellBiotechnology (M. Butler ed., 1991); Animal Cell Culture (J. Pollard etal., eds., Humana Press 1990); Culture of Animal Cells, 2^(nd) Ed. (R.Freshney et al., eds., Alan R. Liss 1987); Flow Cytometry and Sorting(M. Melamed et al., eds., Wiley-Liss 1990); the series Methods inEnzymology (Academic Press, Inc.); Wirth M. and Hauser H. (1993);Immunochemistry in Practice, 3rd edition, A. Johnstone & R. Thorpe,Blackwell Science, Cambridge, Mass., 1996; Techniques inImmunocytochemistry, (G. Bullock & P. Petrusz eds., Academic Press 1982,1983, 1985, 1989); Handbook of Experimental Immunology, (D. Weir & C.Blackwell, eds.); Current Protocols in Immunology (J. Coligan et al.,eds. 1991); Immunoassay (E. P. Diamandis & T. K. Christopoulos, eds.,Academic Press, Inc., 1996); Goding (1986) Monoclonal Antibodies:Principles and Practice (2d ed) Academic Press, New York; Ed Harlow andDavid Lane, Antibodies A laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1988; Antibody Engineering, 2^(nd)edition (C. Borrebaeck, ed., Oxford University Press, 1995); and theseries Annual Review of Immunology; the series Advances in Immunology.

1. Recombinant Production of Proteins in Mammalian Host Cells Using aGlutamine-Free Cell Culture Medium

The present invention concerns the large-scale recombinant production ofproteins in mammalian host cells, using a glutamine-free cell culturemedium supplemented with asparagine. Mammalian cells have become thedominant system for the production of mammalian proteins for clinicalapplications, primarily due to their ability to produce properly foldedand assembled heterologous proteins, and their capacity forpost-translational modifications. Chinese hamster ovary (CHO) cells, andcell lines obtained from various other mammalian sources, such as, forexample, mouse myeloma (NS0), baby hamster kidney (BHK), human embryonickidney (HEK-293) and human retinal cells have been approved byregulatory agencies for the production of biopharmaceutical products,including therapeutic antibodies. Of these, Chinese Hamster Ovary Cells(CHO) are among the most commonly used industrial hosts, which arewidely employed for the production of heterologous proteins. Thus,methods for the large-scale production of antibodies in CHO, includingdihydrofolate reductase negative (DHFR−) CHO cells, are well known inthe art (see, e.g. Trill et al., Curr. Opin. Biotechnol. 6(5):553-60(1995) and U.S. Pat. No. 6,610,516).

As a first step, the nucleic acid (e.g., cDNA or genomic DNA) encodingthe desired recombinant protein may be inserted into a replicable vectorfor cloning (amplification of the DNA) or for expression. Variousvectors are publicly available. The vector components generally include,but are not limited to, one or more of the following: a signal sequence,an origin of replication, one or more marker genes, an enhancer element,a promoter, and a transcription termination sequence, each of which isdescribed below. Optional signal sequences, origins of replication,marker genes, enhancer elements and transcription terminator sequencesthat may be employed are known in the art and described in furtherdetail in PCT Publication WO 97/25428.

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to theprotein-encoding nucleic acid sequence. Promoters are untranslatedsequences located upstream (5′) to the start codon of a structural gene(generally within about 100 to 1000 bp) that control the transcriptionand translation of a particular nucleic acid sequence to which they areoperably linked. Such promoters typically fall into two classes,inducible and constitutive. Inducible promoters are promoters thatinitiate increased levels of transcription from DNA under their controlin response to some change in culture conditions, e.g., the presence orabsence of a nutrient or a change in temperature. At this time a largenumber of promoters recognized by a variety of potential host cells arewell known. These promoters are operably linked to DNA encoding thedesired protein by removing the promoter from the source DNA byrestriction enzyme digestion and inserting the isolated promotersequence into the vector.

Promoters suitable for use with prokaryotic and eukaryotic hosts areknown in the art, and are described in further detail in PCT PublicationNo. WO97/25428.

Construction of suitable vectors containing one or more of theabove-listed components employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and re-ligated in theform desired to generate the plasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures can be used to transform E. coli cells, such as E.coli K12 strain 294 (ATCC® 31,446) and successful transformants selectedby ampicillin or tetracycline resistance where appropriate. Plasmidsfrom the transformants are prepared, analyzed by restrictionendonuclease digestion, and/or sequenced using standard techniques knownin the art. (See, e.g., Messing et al., Nucleic Acids Res. 1981, 9:309;Maxam et al., Methods in Enzymology 1980, 65:499).

Expression vectors that provide for the transient expression inmammalian cells may be employed. In general, transient expressioninvolves the use of an expression vector that is able to replicateefficiently in a host cell, such that the host cell accumulates manycopies of the expression vector and, in turn, synthesizes high levels ofa desired polypeptide encoded by the expression vector (Sambrook et al.,supra). Transient expression systems, comprising a suitable expressionvector and a host cell, allow for the convenient positive identificationof polypeptides encoded by cloned DNAs, as well as for the rapidscreening of such polypeptides for desired biological or physiologicalproperties.

Other methods, vectors, and host cells suitable for adaptation to thesynthesis of a desired heterologous protein in recombinant vertebratecell culture are described in Gething et al., Nature 1981, 293:620-625;Mantei et al., Nature 1979, 281:40-46; EP 117,060; and EP 117,058.

For large-scale production, according to the present invention mammalianhost cells are transfected and preferably transformed with theabove-described expression vectors and cultured in nutrient mediamodified as appropriate for inducing promoters, selecting transformants,or amplifying the genes encoding the desired sequences.

Transfection refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ and electroporation. Successful transfection is generallyrecognized when any indication of the operation of this vector occurswithin the host cell.

Transformation means introducing DNA into an organism so that the DNA isreplicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. The calcium treatmentemploying calcium chloride, as described in Sambrook et al., supra, orelectroporation is generally used for prokaryotes or other cells thatcontain substantial cell-wall barriers. Infection with Agrobacteriumtumefaciens is used for transformation of certain plant cells, asdescribed (Shaw et al., Gene 1983, 23:315 and PCT Publication No. WO89/05859). In addition, plants may be transfected using ultrasoundtreatment, PCT Publication No. WO 91/00358 published 10 Jan. 1991.

For mammalian cells without such cell walls, the calcium phosphateprecipitation method (Graham and van der Eb, Virology 1978, 52:456-457)may be employed. General aspects of mammalian cell host systemtransformations have been described in U.S. Pat. No. 4,399,216. Forvarious techniques for transforming mammalian cells, see also Keown etal. Methods in Enzymology 1990, 185:527-537 and Mansour et al. Nature1988, 336:348-352.

During large-scale production, to begin the production cycle usually asmall number of transformed recombinant host cells is allowed to grow inculture for several days. Once the cells have undergone several roundsof replication, they are transferred to a larger container where theyare prepared to undergo fermentation. The media in which the cells aregrown and the levels of oxygen, nitrogen and carbon dioxide that existduring the production cycle may have a significant impact on theproduction process. Growth parameters are determined specifically foreach cell line and these parameters are measured frequently to assureoptimal growth and production conditions.

When the cells grow to sufficient numbers, they are transferred tolarge-scale production tanks to begin the production phase, and grownfor a longer period of time. At this point in the process, therecombinant protein can be harvested. Typically, the cells areengineered to secrete the polypeptide into the cell culture media, sothe first step in the purification process is to separate the cells fromthe media. Harvesting usually includes centrifugation and filtration toproduce a Harvested Cell Culture Fluid (HCCF). The media is thensubjected to several additional purification steps that remove anycellular debris, unwanted proteins, salts, minerals or other undesirableelements. At the end of the purification process, the recombinantprotein is highly pure and is suitable for human therapeutic use.

Although this process has been the subject of much study andimprovements over the past several decades, there is room fur furtherimprovements in the large-scale commercial production of recombinantproteins, such as antibodies. Thus, increases in cell viability,longevity and specific productivity of mammalian host cell cultures, andimprovements in the titer of the recombinant proteins produced have agenuine impact on the price of the recombinant protein produced, and, inthe case of therapeutic proteins, the price and availability of drugproducts.

The present invention concerns an improved method for the production ofheterologous proteins in mammalian cell culture, using a glutamine-freeculture medium with added asparagine in the production phase of the cellculture process. The culture media used in the process of the presentinvention can be based on any commercially available medium forrecombinant production of proteins in mammalian host cells, inparticular CHO cells.

Examples of commercially available culture media include Ham's F10(Sigma), Minimal Essential Medium (“MEM”, Sigma), RPMI-1640 (Sigma), andDulbecco's Modified Eagle's Medium (“DMEM”, Sigma). Any such media maybe supplemented as necessary with hormones and/or other growth factors(such as insulin, transferrin, or epidermal growth factor), salts (suchas sodium chloride, calcium, magnesium, and phosphate), buffers (such asHEPES), nucleosides (such as adenosine and thymidine), antibiotics (suchas Gentamycin™ drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan. In addition, the culture media of the present inventioncan be based any of the media described in Ham and McKeehan, Meth. Enz.,58: 44 (1979); Barnes and Sato, Anal. Biochem., 102: 255 (1980); U.S.Pat. No. 4,767,704; U.S. Pat. No. 4,657,866; U.S. Pat. No. 4,927,762;U.S. Pat. No. 5,122,469 or U.S. Pat. No. 4,560,655; WO 90/03430; and WO87/00195, provided that glutamine is omitted as an ingredient.

Under Glutamine-free conditions Asparagine is required since mammaliancells can synthesize Asparagine only in presence of Glutamine.Asparagine is synthesized by amide transfer from Glutamine in thepresence of Asparagine synthetase. The Asparagine is preferably added tothe culture medium at a concentration in the range of 2.5 mM to 15 mM.In various embodiments of the present invention, the preferredconcentration of Asparagine should be at least 2.5 mM. In preferredembodiments, the asparagine is added at a concentration of 10 mM.

In general, principles, protocols, and practical techniques formaximizing the productivity of mammalian cell cultures can be found in,and can be adapted for the production of recombinant proteins using thecell culture media herein.

The necessary nutrients and growth factors for the medium, includingtheir concentrations, for a particular cell line, are determinedempirically without undue experimentation as described, for example, inMammalian Cell Culture, Mather, ed. (Plenum Press: NY, 1984); Barnes andSato, Cell, 22: 649 (1980) or Mammalian Cell Biotechnology: A PracticalApproach, M. Butler, ed. (IRL Press, 1991). A suitable medium contains abasal medium component such as a DMEM/HAM F-12-based formulation (forcomposition of DMEM and HAM F12 media and especially serum-free media,see culture media formulations in American Type Culture CollectionCatalogue of Cell Lines and Hybridomas, Sixth Edition, 1988, pages346-349), with modified concentrations of some components such as aminoacids, salts, sugar, and vitamins, and optionally containing glycine,hypoxanthine, and thymidine; recombinant human insulin, hydrolyzedpeptone, such as PRIMATONE HS™ or PRIMATONE RL™ (Sheffield, England), orthe equivalent; a cell protective agent, such as PLURONIC F68™ or theequivalent pluronic polyol; GENTAMYCIN™; and trace elements. Theformulations of medium as described in U.S. Pat. No. 5,122,469,characterized by the presence of high levels of certain amino acids, aswell as PS-20 as described below, are particularly appropriate.

The glycoproteins of the present invention may be produced by growingcells which express the desired glycoprotein under a variety of cellculture conditions. For instance, cell culture procedures for the large-or small-scale production of glycoproteins are potentially useful withinthe context of the present invention. Procedures including, but notlimited to, a fluidized bed bioreactor, hollow fiber bioreactor, rollerbottle culture, or stirred tank bioreactor system may be used, in thelater two systems, with or without microcarriers, and operatedalternatively in a batch, fed-batch, or continuous mode.

In a particular embodiment the cell culture of the present invention isperformed in a stirred tank bioreactor system and a fed-batch cultureprocedure is employed. In the preferred fed-batch culture the mammalianhost cells and culture medium are supplied to a culturing vesselinitially and additional culture nutrients are fed, continuously or indiscrete increments, to the culture during culturing, with or withoutperiodic cell and/or product harvest before termination of culture. Thefed-batch culture can include, for example, a semi-continuous fed-batchculture, wherein periodically whole culture (including cells and medium)is removed and replaced by fresh medium Fed-batch culture isdistinguished from simple-batch culture in which all components for cellculturing (including the cells and all culture nutrients) are suppliedto the culturing vessel at the start of the culturing process. Fed-batchculture can be further distinguished from perfusion culturing insofar asthe supernate is not removed from the culturing vessel during theprocess (in perfusion culturing, the cells are restrained in the cultureby, e.g., filtration, encapsulation, anchoring to microcarriers, etc.,and the culture medium is continuously or intermittently introduced andremoved from the culturing vessel).

Further, the cells of the culture may be propagated according to anyscheme or routine that may be suitable for the particular host cell andthe particular production plan contemplated. Therefore, the presentinvention contemplates a single-step or multiple-step culture procedure.In a single-step culture the host cells are inoculated into a cultureenvironment and the processes of the instant invention are employedduring a single production phase of the cell culture. Alternatively, amulti-stage culture is envisioned. In the multi-stage culture cells maybe cultivated in a number of steps or phases. For instance, cells may begrown in a first step or growth phase culture wherein cells, possiblyremoved from storage, are inoculated into a medium suitable forpromoting growth and high viability. The cells may be maintained in thegrowth phase for a suitable period of time by the addition of freshmedium to the host cell culture.

According to a specific aspect of the invention, fed-batch or continuouscell culture conditions are devised to enhance growth of the mammaliancells in the growth phase of the cell culture. In the growth phase cellsare grown under conditions and for a period of time that is maximizedfor growth. Culture conditions, such as temperature, pH, dissolvedoxygen (DO₂), and the like, are those used with the particular host andwill be apparent to the ordinarily-skilled artisan. Generally, the pH isadjusted to a level between about 6.5 and 7.5 using either an acid(e.g., CO₂) or a base (e.g., Na₂CO₃ or NaOH). A suitable temperaturerange for culturing mammalian cells such as CHO cells is between about30 to 40° C. and preferably about 37° C. and a suitable DO₂ is between5-90% of air saturation.

At a particular stage the cells may be used to inoculate a productionphase or step of the cell culture. Alternatively, as described above theproduction phase or step may be continuous with the inoculation orgrowth phase or step.

Production of a target protein in mammalian, e.g., CHO, cells typicallyemploys a semi-continuous process whereby cells are culture in a“seed-train” for various periods of time and are periodicallytransferred to inoculum fermentors to generate enough cell mass toinoculate a production fermentor at larger scale. Thus, cells used forthe production of the desired protein are in culture for various periodsof time up to a maximum predefined cell age. The parameters of the cellculture process, such as seed density, pH, DO₂ and temperature duringculture, duration of the production culture, operating conditions ofharvest, etc. are a function of the particular cell line and culturemedium used, and can be determined empirically, without undueexperimentation.

According to the present invention, the cell-culture environment duringthe production phase of the cell culture is controlled. In a preferredaspect, the production phase of the cell culture process is preceded bya transition phase of the cell culture in which parameters for theproduction phase of the cell culture are engaged.

The desired polypeptide, such as antibody, preferably is recovered fromthe culture medium as a secreted polypeptide, although it also may berecovered from host cell lysates when directly produced without asecretory signal. If the polypeptide is membrane-bound, it can bereleased from the membrane using a suitable detergent solution (e.g.,Triton-X 100) or its extracellular region may be released by enzymaticcleavage.

When the polypeptide is produced in a recombinant cell other than one ofhuman origin, it is free of proteins or polypeptides of human origin.However, it is usually necessary to recover or purify recombinantproteins from recombinant cell proteins or polypeptides to obtainpreparations that are substantially homogeneous as to the desiredpolypeptide. As a first step, the culture medium or lysate may becentrifuged to remove particulate cell debris. The heterologouspolypeptide thereafter is purified from contaminant soluble proteins andpolypeptides, with the following procedures being exemplary of suitablepurification procedures: by fractionation on an ion-exchange column suchas SP-Sepharose™ or CM-Sepharose™; hydroxyapatite; hydrophobicinteraction chromatography; ethanol precipitation;

chromatofocusing; ammonium sulfate precipitation; gel filtration using,for example, Sephadex G-75™; and/or diafiltration.

Recombinant polypeptides can be isolated, e.g. by affinitychromatography.

A protease inhibitor such as phenyl methyl sulfonyl fluoride (PMSF) alsomay be useful to inhibit proteolytic degradation during purification,and antibiotics may be included to prevent the growth of adventitiouscontaminants. One skilled in the art will appreciate that purificationmethods suitable for the purification and isolation of recombinantproteins, including antibodies, can be used herein, and modified ifneeded, using standard techniques.

Expression of the desired heterologous protein may be measured in asample directly, for example, by conventional Southern blotting,Northern blotting to quantitate the transcription of mRNA (Thomas, Proc.Natl. Acad. Sci. USA 1980, 77:5201-5205), dot blotting (DNA analysis),or in situ hybridization, using an appropriately labeled probe, based onthe sequences provided herein. Various labels may be employed, mostcommonly radioisotopes, and particularly ³²P. However, other techniquesmay also be employed, such as using biotin-modified nucleotides forintroduction into a polynucleotide. The biotin then serves as the sitefor binding to avidin or antibodies, which may be labeled with a widevariety of labels, such as radionucleotides, fluorescers or enzymes.Alternatively, antibodies may be employed that can recognize specificduplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybridduplexes or DNA-protein duplexes. The antibodies in turn may be labeledand the assay may be carried out where the duplex is bound to a surface,so that upon the formation of duplex on the surface, the presence ofantibody bound to the duplex can be detected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. With immunohistochemicalstaining techniques, a cell sample is prepared, typically by dehydrationand fixation, followed by reaction with labeled antibodies specific forthe gene product coupled, where the labels are usually visuallydetectable, such as enzymatic labels, fluorescent labels, luminescentlabels, and the like. Antibodies useful for immunohistochemical stainingand/or assay of sample fluids may be either monoclonal or polyclonal,and may be prepared in any mammal

2. Antibodies

In a preferred embodiment, the methods of the present invention are usedfor the recombinant production of antibodies, including therapeutic anddiagnostic antibodies. Antibodies within the scope of the presentinvention include, but are not limited to: anti-HER2 antibodiesincluding Trastuzumab (HERCEPTIN®) (Carter et al., Proc. Natl. Acad.Sci. USA, 89:4285-4289 (1992), U.S. Pat. No. 5,725,856); anti-CD20antibodies such as chimeric anti-CD20 “C2B8” as in U.S. Pat. No.5,736,137 (RITUXAN®), a chimeric or humanized variant of the 2H7antibody as in U.S. Pat. No. 5,721,108B1, or Tositumomab (BEXXAR®);anti-IL-8 (St John et al., Chest, 103:932 (1993), and InternationalPublication No. WO 95/23865); anti-VEGF antibodies including humanizedand/or affinity matured anti-VEGF antibodies such as the humanizedanti-VEGF antibody huA4.6.1 AVASTIN® (Kim et al., Growth Factors,7:53-64 (1992), International Publication No. WO 96/30046, and WO98/45331, published Oct. 15, 1998); anti-PSCA antibodies (WO01/40309);anti-CD40 antibodies, including S2C6 and humanized variants thereof(WO00/75348); anti-CD11a (U.S. Pat. No. 5,622,700, WO 98/23761, Steppeet al., Transplant Intl. 4:3-7 (1991), and Hourmant et al.,Transplantation 58:377-380 (1994)); anti-IgE (Presta et al., J. Immunol.151:2623-2632 (1993), and International Publication No. WO 95/19181);anti-CD18 (U.S. Pat. No. 5,622,700, issued Apr. 22, 1997, or as in WO97/26912, published Jul. 31, 1997); anti-IgE (including E25, E26 andE27; U.S. Pat. No. 5,714,338, issued Feb. 3, 1998 or U.S. Pat. No.5,091,313, issued Feb. 25, 1992, WO 93/04173 published Mar. 4, 1993, orInternational Application No. PCT/US98/13410 filed Jun. 30, 1998, U.S.Pat. No. 5,714,338); anti-Apo-2 receptor antibody (WO 98/51793 publishedNov. 19, 1998); anti-TNF-α antibodies including cA2 (REMICADE®), CDP571and MAK-195 (See, U.S. Pat. No. 5,672,347 issued Sep. 30, 1997, Lorenzet al., J. Immunol. 156(4):1646-1653 (1996), and Dhainaut et al., Crit.Care Med. 23(9):1461-1469 (1995)); anti-Tissue Factor (TF) (EuropeanPatent No. 0 420 937 B1 granted Nov. 9, 1994); anti-human α₄β₇ integrin(WO 98/06248 published Feb. 19, 1998); anti-EGFR (chimerized orhumanized 225 antibody as in WO 96/40210 published Dec. 19, 1996);anti-CD3 antibodies such as OKT3 (U.S. Pat. No. 4,515,893 issued May 7,1985); anti-CD25 or anti-tac antibodies such as CHI-621 (SIMULECT®) and(ZENAPAX®) (See U.S. Pat. No. 5,693,762 issued Dec. 2, 1997); anti-CD4antibodies such as the cM-7412 antibody (Choy et al., Arthritis Rheum39(1):52-56 (1996)); anti-CD52 antibodies such as CAMPATH-1H (Riechmannet al., Nature 332:323-337 (1988)); anti-Fc receptor antibodies such asthe M22 antibody directed against FcγRI as in Graziano et al., J.Immunol. 155(10):4996-5002 (1995); anti-carcinoembryonic antigen (CEA)antibodies such as hMN-14 (Sharkey et al., Cancer Res. 55(23Suppl):5935s-5945s (1995); antibodies directed against breast epithelial cellsincluding huBrE-3, hu-Mc 3 and CHL6 (Ceriani et al., Cancer Res. 55(23):5852s-5856s (1995); and Richman et al., Cancer Res. 55(23 Supp):5916s-5920s (1995)); antibodies that bind to colon carcinoma cells suchas C242 (Litton et al., Eur J. Immunol. 26(1):1-9 (1996)); anti-CD38antibodies, e.g. AT 13/5 (Ellis et al., J. Immunol. 155(2):925-937(1995)); anti-CD33 antibodies such as Hu M195 (Jurcic et al., Cancer Res55(23 Suppl):5908s-5910s (1995) and CMA-676 or CDP771; anti-CD22antibodies such as LL2 or LymphoCide (Juweid et al., Cancer Res 55(23Suppl):5899s-5907s (1995)); anti-EpCAM antibodies such as 17-1A(PANOREX®); anti-GpIIb/IIIa antibodies such as abciximab or c7E3 Fab(REOPRO®); anti-RSV antibodies such as MEDI-493 (SYNAGIS®); anti-CMVantibodies such as PROTOVIR®; anti-HIV antibodies such as PRO542;anti-hepatitis antibodies such as the anti-Hep B antibody OSTAVIR®;anti-CA 125 antibody OvaRex; anti-idiotypic GD3 epitope antibody BEC2;anti-αvβ3 antibody VITAXIN®; anti-human renal cell carcinoma antibodysuch as ch-G250; ING-1; anti-human 17-1A antibody (3622W94); anti-humancolorectal tumor antibody (A33); anti-human melanoma antibody R24directed against GD3 ganglioside; anti-human squamous-cell carcinoma(SF-25); and anti-human leukocyte antigen (HLA) antibodies such as SmartID10 and the anti-HLA DR antibody Oncolym (Lym-1). The preferred targetantigens for the antibody herein are: HER2 receptor, VEGF, IgE, CD20,CD11a, and CD40.

Many of these antibodies are widely used in clinical practice to treatvarious diseases, including cancer.

In certain specific embodiments, the methods of the present inventionare used for the production of the following antibodies and recombinantproteins.

Anti-CD20 Antibodies

Rituximab (RITUXAN®) is a genetically engineered chimeric murine/humanmonoclonal antibody directed against the CD20 antigen. Rituximab is theantibody called “C2B8” in U.S. Pat. No. 5,736,137 issued Apr. 7, 1998(Anderson et al.). Rituximab is indicated for the treatment of patientswith relapsed or refractory low-grade or follicular, CD20-positive, Bcell non-Hodgkin's lymphoma. In vitro mechanism of action studies havedemonstrated that rituximab binds human complement and lyses lymphoid Bcell lines through complement-dependent cytotoxicity (CDC) (Reff et al.,Blood 83(2):435-445 (1994)). Additionally, it has significant activityin assays for antibody-dependent cellular cytotoxicity (ADCC). Morerecently, rituximab has been shown to have anti-proliferative effects intritiated thymidine incorporation assays and to induce apoptosisdirectly, while other anti-CD19 and CD20 antibodies do not (Maloney etal., Blood 88(10):637a (1996)). Synergy between rituximab andchemotherapies and toxins has also been observed experimentally. Inparticular, rituximab. sensitizes drug-resistant human B cell lymphomacell lines to the cytotoxic effects of doxorubicin, CDDP, VP-1 6,diphtheria toxin and ricin (Demidem et al., Cancer Chemotherapy &Radiopharmaceuticals 12(3):177-186 (1997)). In vivo preclinical studieshave shown that rituximab depletes B cells from the peripheral blood,lymph nodes, and bone marrow of cynomolgus monkeys, presumably throughcomplement and cell-mediated processes (Reff et al., Blood 83(2):435-445(1994)).

Patents and patent publications concerning CD20 antibodies include U.S.Pat. Nos. 5,776,456, 5,736,137, 6,399,061, and 5,843,439, as well asU.S. patent application Nos. US 2002/0197255A1, US 2003/0021781A1, US2003/0082172 A1, US 2003/0095963 A1, US 2003/0147885 A1 (Anderson etal.); U.S. Pat. No. 6,455,043B1 and WO00/09160 (Grillo-Lopez, A.);WO00/27428 (Grillo-Lopez and White); WO00/27433 (Grillo-Lopez andLeonard); WO00/44788 (Braslawsky et al.); WO01/10462 (Rastetter, W.);WO01/10461 (Rastetter and White); WO01/10460 (White and Grillo-Lopez);U.S. application No. US2002/0006404 and WO02/04021 (Hanna andHariharan); U.S. application No. US2002/0012665 A1 and WO01/74388(Hanna, N.); U.S. application No. US 2002/0058029 A1 (Hanna, N.); U.S.application No. US 2003/0103971 A1 (Hariharan and Hanna); U.S.application No. US2002/0009444A1, and WO01/80884 (Grillo-Lopez, A.);WO01/97858 (White, C.); U.S. application No. US2002/0128488A1 andWO02/34790 (Reff, M.); W)02/060955 (Braslawsky et al.); WO2/096948(Braslawsky et al.); WO02/079255 (Reff and Davies); U.S. Pat. No.6,171,586B1, and WO98/56418 (Lam et al.); WO98/58964 (Raju, S.);WO99/22764 (Raju, S.); WO99/51642, U.S. Pat. No. 6,194,551B1, U.S. Pat.No. 6,242,195B1, U.S. Pat. No. 6,528,624B1 and U.S. Pat. No. 6,538,124(Idusogie et al.); WO00/42072 (Presta, L.); WO00/67796 (Curd et al.);WO01/03734 (Grillo-Lopez et al.); U.S. application No. US 2002/0004587A1and WO01/77342 (Miller and Presta); U.S. application No. US2002/0197256(Grewal, I.); U.S. application No. US 2003/0157108 A1 (Presta, L.); U.S.Pat. Nos. 6,090,365B1, 6,287,537B1, 6,015,542, 5,843,398, and 5,595,721,(Kaminski et al.); U.S. Pat. Nos. 5,500,362, 5,677,180, 5,721,108, and6,120,767 (Robinson et al.); U.S. Pat. No. 6,410,391B1 (Raubitschek etal.); U.S. Pat. No. 6,224,866B1 and WO00/20864 (Barbera-Guillem, E.);WO01/13945 (Barbera-Guillem, E.); WO00/67795 (Goldenberg); U.S.application No. US 2003/01339301 A1 and WO00/74718 (Goldenberg andHansen); WO00/76542 (Golay et al.); WO01/72333 (Wolin and Rosenblatt);U.S. Pat. No. 6,368,596B1 (Ghetie et al.); U.S. application No.US2002/0041847 A1, (Goldenberg, D.); U.S. application No.US2003/0026801A1 (Weiner and Hartmann); WO02/102312 (Engleman, E.); U.S.patent application No. 2003/0068664 (Albitar et al.); WO03/002607(Leung, S.); WO 03/049694 and US 2003/0185796 A1 (Wolin et al.);WO03/061694 (Sing and Siegall); US 2003/0219818 A1 (Bohen et al.); US2003/0219433 A1 and WO 03/068821 (Hansen et al.) each of which isexpressly incorporated herein by reference. See, also, U.S. Pat. No.5,849,898 and EP application no. 330,191 (Seed et al.); U.S. Pat. No.4,861,579 and EP332,865A2 (Meyer and Weiss); U.S. Pat. No. 4,861,579(Meyer et al.) and WO95/03770 (Bhat et al.).

Publications concerning therapy with Rituximab include: Perotta andAbuel “Response of chronic relapsing ITP of 10 years duration toRituximab” Abstract #3360 Blood 10(1)(part 1-2): p. 88B (1998); Stashiet al., “Rituximab chimeric anti-CD20 monoclonal antibody treatment foradults with chronic idopathic thrombocytopenic purpura” Blood98(4):952-957 (2001); Matthews, R. “Medical Heretics” New Scientist (7Apr. 2001); Leandro et al., “Clinical outcome in 22 patients withrheumatoid arthritis treated with B lymphocyte depletion” Ann Rheum Dis61:833-888 (2002); Leandro et al., “Lymphocyte depletion in rheumatoidarthritis: early evidence for safety, efficacy and dose response.Arthritis & Rheumatism 44(9): 5370 (2001); Leandro et al., “An openstudy of B lymphocyte depletion in systemic lupus erythematosus”,Arthritis & Rheumatism 46(1):2673-2677 (2002); Edwards and Cambridge“Sustained improvement in rheumatoid arthritis following a protocoldesigned to deplete B lymphocytes” Rheumatology 40:205-211 (2001);Edwards et al., “B-lymphocyte depletion therapy in rheumatoid arthritisand other autoimmune disorders” Biochem. Soc. Trans. 30(4):824-828(2002); Edwards et al., “Efficacy and safety of Rituximab, a B-celltargeted chimeric monoclonal antibody: A randomized, placebo controlledtrial in patients with rheumatoid arthritis. Arthritis & Rheumatism46(9): 5197 (2002); Levine and Pestronk “IgM antibody-relatedpolyneuropathies: B-cell depletion chemotherapy using Rituximab”Neurology 52: 1701-1704 (1999); DeVita et al., “Efficacy of selective Bcell blockade in the treatment of rheumatoid arthritis” Arthritis &Rheumatism 46:2029-2033 (2002); Hidashida et al., “Treatment ofDMARD-Refractory rheumatoid arthritis with rituximab.” Presented at theAnnual Scientific Meeting of the American College of Rheumatology;October 24-29; New Orleans, La. 2002; Tuscano, J. “Successful treatmentof Infliximab-refractory rheumatoid arthritis with rituximab” Presentedat the Annual Scientific Meeting of the American College ofRheumatology; October 24-29; New Orleans, La. 2002. Sarwal et al., N.Eng. J. Med. 349(2):125-138 (Jul. 10, 2003) reports molecularheterogeneity in acute renal allograft rejection identified by DNAmicroarray profiling.

In various embodiments, the invention provides pharmaceuticalcompositions comprising humanized anti-CD20 antibodies. In certainembodiments, the humanized antibody composition of the invention furthercomprises amino acid alterations in the IgG Fc and exhibits increasedbinding affinity for human FcRn over an antibody having wild-type IgGFc, by at least 60 fold, at least 70 fold, at least 80 fold, morepreferably at least 100 fold, preferably at least 125 fold, even morepreferably at least 150 fold to about 170 fold.

The N-glycosylation site in IgG is at Asn297 in the C_(H)2 domain.Humanized antibody compositions of the present invention includecompositions of any of the preceding humanized antibodies having an Fcregion, wherein about 80-100% (and preferably about 90-99%) of theantibody in the composition comprises a mature core carbohydratestructure which lacks fucose, attached to the Fc region of theglycoprotein. Such compositions were demonstrated herein to exhibit asurprising improvement in binding to Fc(RIIIA(F158), which is not aseffective as Fc(RIIIA (V158) in interacting with human IgG. Fc(RIIIA(F158) is more common than Fc(RIIIA (V 158) in normal, healthy AfricanAmericans and Caucasians. See Lehrnbecher et al., Blood 94:4220 (1999).Historically, antibodies produced in Chinese Hamster Ovary Cells (CHO),one of the most commonly used industrial hosts, contain about 2 to 6% inthe population that are nonfucosylated. YB2/0 and Lec13, however, canproduce antibodies with 78 to 98% nonfucosylated species. Shinkawa etal., J Bio. Chem. 278 (5), 3466-347 (2003), reported that antibodiesproduced in YB2/0 and Lec13 cells, which have less FUT8 activity, showsignificantly increased ADCC activity in vitro. The production ofantibodies with reduced fucose content are also described in e.g., Li etal., (GlycoFi) “Optimization of humanized IgGs in glycoengineered Pichiapastoris” in Nature Biology online publication 22 Jan. 2006; Niwa R. etal., Cancer Res. 64(6):2127-2133 (2004); US 2003/0157108 (Presta); U.S.Pat. No. 6,602,684 and US 2003/0175884 (Glycart Biotechnology); US2004/0093621, US 2004/0110704, US 2004/0132140 (all of Kyowa HakkoKogyo).

A bispecific humanized antibody encompasses an antibody wherein one armof the antibody has at least the antigen binding region of the H and/orL chain of a humanized antibody of the invention, and the other arm hasV region binding specificity for a second antigen. In specificembodiments, the antigens are selected from the group consisting ofCD-20, CD3, CD64, CD32A, CD16, NKG2D or other NK activating ligands.

Anti-HER2 Antibodies

A recombinant humanized version of the murine HER2 antibody 4D5(huMAb4D5-8, rhuMAb HER2, trastuzumab or HERCEPTIN®; U.S. Pat. No.5,821,337) is clinically active in patients with HER2-overexpressingmetastatic breast cancers that have received extensive prior anti-cancertherapy (Baselga et al., J. Clin. Oncol. 14:737-744 (1996)). Trastuzumabreceived marketing approval from the Food and Drug Administration (FDA)Sep. 25, 1998 for the treatment of patients with metastatic breastcancer whose tumors overexpress the HER2 protein. In November 2006, theFDA approved Herceptin as part of a treatment regimen containingdoxorubicin, cyclophosphamide and paclitaxel, for the adjuvant treatmentof patients with HER2-positive, node-positive breast cancer.

In various embodiments, the invention provides pharmaceuticalcompositions comprising humanized anti-HER2 antibodies. HER2 antibodieswith various properties have been described in Tagliabue et al., Int. J.Cancer 47:933-937 (1991); McKenzie et al., Oncogene 4:543-548 (1989);Maier et al., Cancer Res. 51:5361-5369 (1991); Bacus et al., MolecularCarcinogenesis 3:350-362 (1990); Stancovski et al., PNAS (USA)88:8691-8695 (1991); Bacus et al., Cancer Research 52:2580-2589 (1992);Xu et al., Int. J. Cancer 53:401-408 (1993); WO94/00136; Kasprzyk etal., Cancer Research 52:2771-2776 (1992); Hancock et al., Cancer Res.51:4575-4580 (1991); Shawver et al., Cancer Res. 54:1367-1373 (1994);Arteaga et al., Cancer Res. 54:3758-3765 (1994); Harwerth et al., J.Biol. Chem. 267:15160-15167 (1992); U.S. Pat. No. 5,783,186; and Klapperet al., Oncogene 14:2099-2109 (1997).

Anti-VEGF Antibodies

anti-VEGF antibodies including humanized and/or affinity maturedanti-VEGF antibodies such as the humanized anti-VEGF antibody huA4.6.1AVASTIN® (Kim et al., Growth Factors, 7:53-64 (1992), InternationalPublication No. WO 96/30046, and WO 98/45331, published Oct. 15, 1998)are FDA approved for the treatment of cancer. In various embodiments,the invention provides pharmaceutical compositions comprising humanizedanti-VEGF antibodies.

Anti-CD11a Antibodies

The humanized anti-CD11a antibody efalizumab or Raptiva (U.S. Pat. No.6,037,454) received marketing approval from the Food and DrugAdministration on Oct. 27, 2003 for the treatment for the treatment ofpsoriasis. One embodiment provides for pharmaceutical compositionscomprising anti-human CD11a antibodies.

Apomab Antibodies

Antibodies to the DR5 receptor (anti-DR5) antibodies can also beproduced in accordance with the present invention. Such anti-DR5antibodies specifically include all antibody variants disclosed in PCTPublication No. WO 2006/083971, such as the anti-DR5 antibodiesdesignated Apomabs 1.1, 2.1, 3.1, 4.1, 5.1, 5.2, 5.3, 6.1, 6.2, 6.3,7.1, 7.2, 7.3, 8.1, 8.3, 9.1, 1.2, 2.2, 3.2, 4.2, 5.2, 6.2, 7.2, 8.2,9.2, 1.3, 2.2, 3.3, 4.3, 5.3, 6.3, 7.3, 8.3, 9.3, and 25.3, especiallyApomab 8.3 and Apomab 7.3, preferably Apomab 7.3. The entire content ofWO 2006/083971 is hereby expressly incorporated by reference. Apomab isa fully human monoclonal antibody which is a DR5-targeted pro-apoptoticreceptor agonist (PARA) specifically designed to induce apoptosis.Apoptosis is a natural process by which damaged or unwanted cells,including those that are cancerous, die and are cleared from the body.Pro-apoptotic receptor DR5 is expressed in a broad range ofmalignancies.

Anti-BR3 Antibodies and Immunoadhesins

Antibodies to the BR3 (anti-BR3) antibodies and BR3-Fc immunoadhesinscan also be produced in accordance with the present invention. Suchanti-BR3 antibodies and immunoadhesins specifically include all variantsdisclosed in U.S. Application Publication No. 20050070689. The entirecontent of U.S. Application Publication No. 20050070689 is herebyexpressly incorporated by reference.

3. General Methods for the Recombinant Production of Antibodies

The antibodies and other recombinant proteins herein can be produced bywell known techniques of recombinant DNA technology. Thus, aside fromthe antibodies specifically identified above, the skilled practitionercould generate antibodies directed against an antigen of interest, e.g.,using the techniques described below.

Antigen Selection and Preparation

The antibody herein is directed against an antigen of interest.Preferably, the antigen is a biologically important polypeptide andadministration of the antibody to a mammal suffering from a disease ordisorder can result in a therapeutic benefit in that mammal However,antibodies directed against nonpolypeptide antigens (such astumor-associated glycolipid antigens; see U.S. Pat. No. 5,091,178) arealso contemplated. Where the antigen is a polypeptide, it may be atransmembrane molecule (e.g. receptor) or ligand such as a growthfactor. Exemplary antigens include those proteins described in section(3) below. Exemplary molecular targets for antibodies encompassed by thepresent invention include CD proteins such as CD3, CD4, CD8, CD19, CD20,CD22, CD34, CD40; members of the ErbB receptor family such as the EGFreceptor, HER2, HER3 or HER4 receptor; cell adhesion molecules such asLFA-1, Mac1, p150,95, VLA-4, ICAM-1, VCAM and αv/β3 integrin includingeither α or β subunits thereof (e.g. anti-CD11a, anti-CD18 or anti-CD11bantibodies); growth factors such as VEGF; IgE; blood group antigens;flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; proteinC, or any of the other antigens mentioned herein. Antigens to which theantibodies listed above bind are specifically included within the scopeherein.

Soluble antigens or fragments thereof, optionally conjugated to othermolecules, can be used as immunogens for generating antibodies. Fortransmembrane molecules, such as receptors, fragments of these (e.g. theextracellular domain of a receptor) can be used as the immunogen.Alternatively, cells expressing the transmembrane molecule can be usedas the immunogen. Such cells can be derived from a natural source (e.g.cancer cell lines) or may be cells which have been transformed byrecombinant techniques to express the transmembrane molecule.

Other antigens and forms thereof useful for preparing antibodies will beapparent to those in the art.

Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the antigen to aprotein that is immunogenic in the species to be immunized, e.g.,keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, whereR and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later the animals are boosted with ⅕to 1/10the originalamount of antigen or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. Preferably, the animal is boostedwith the conjugate of the same antigen, but conjugated to a differentprotein and/or through a different cross-linking reagent. Conjugatesalso can be made in recombinant cell culture as protein fusions. Also,aggregating agents such as alum are suitably used to enhance the immuneresponse.

Monoclonal Antibodies

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster or macaque monkey, is immunized as hereinabove described toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, Protein A-Sepharose, hydroxyapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography. Preferably theProtein A chromatography procedure described herein is used.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the monoclonal antibodies). The hybridoma cells serve asa preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transfected into host cells suchas E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells,or myeloma cells that do not otherwise produce immunoglobulin protein,to obtain the synthesis of monoclonal antibodies in the recombinant hostcells.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, etal., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

In a further embodiment, monoclonal antibodies can be isolated fromantibody phage libraries generated using the techniques described inMcCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991)describe the isolation of murine and human antibodies, respectively,using phage libraries. Subsequent publications describe the productionof high affinity (nM range) human antibodies by chain shuffling (Markset al., Bio/Technology, 10:779-783 (1992)), as well as combinatorialinfection and in vivo recombination as a strategy for constructing verylarge phage libraries (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266(1993)). Thus, these techniques are viable alternatives to traditionalhybridoma techniques for isolation of monoclonal antibodies.

Humanized and Human Antibodies

A humanized antibody has one or more amino acid residues introduced intoit from a source which is non-human. These non-human amino acid residuesare often referred to as “import” residues, which are typically takenfrom an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.,Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman FR for the humanized antibody (Sims et al., J. Immunol., 151:2296(1993)). Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA, 89:4285 (1992); Presta et al., J. Immnol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

Alternatively, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann etal., Year in Immuno., 7:33 (1993); and Duchosal et al., Nature 355:258(1992). Human antibodies can also be derived from phage-displaylibraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks etal., J Mol. Biol., 222:581-597 (1991); Vaughan et al., Nature Biotech14:309 (1996)).

Antibody Fragments

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992) and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. For example, the antibodyfragments can be isolated from the antibody phage libraries discussedabove. Alternatively, Fab′-SH fragments can be directly recovered fromE. coli and chemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163-167 (1992)). According to another approach,F(ab′)₂ fragments can be isolated directly from recombinant host cellculture. Other techniques for the production of antibody fragments willbe apparent to the skilled practitioner. In other embodiments, theantibody of choice is a single chain Fv fragment (scFv) (see WO93/16185).

Multispecific Antibodies

Multispecific antibodies have binding specificities for at least twodifferent antigens. While such molecules normally will only bind twoantigens (i.e. bispecific antibodies, BsAbs), antibodies with additionalspecificities such as trispecific antibodies are encompassed by thisexpression when used herein.

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to another approach described in WO96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the C_(H)3domain of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the ErbB2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol., 152:5368 (1994).Alternatively, the antibodies can be “linear antibodies” as described inZapata et al., Protein Eng. 8(10):1057-1062 (1995). Briefly, theseantibodies comprise a pair of tandem Fd segments(V_(H)-C_(H)1-V_(H)-C_(H)1) which form a pair of antigen bindingregions. Linear antibodies can be bispecific or monospecific.

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147: 60(1991).

Immunoadhesins

The simplest and most straightforward immunoadhesin design combines thebinding domain(s) of the adhesin (e.g. the extracellular domain (ECD) ofa receptor) with the hinge and Fc regions of an immunoglobulin heavychain. Ordinarily, when preparing the immunoadhesins of the presentinvention, nucleic acid encoding the binding domain of the adhesin willbe fused C-terminally to nucleic acid encoding the N-terminus of animmunoglobulin constant domain sequence, however N-terminal fusions arealso possible.

Typically, in such fusions the encoded chimeric polypeptide will retainat least functionally active hinge, C_(H)2 and C_(H)3 domains of theconstant region of an immunoglobulin heavy chain. Fusions are also madeto the C-terminus of the Fc portion of a constant domain, or immediatelyN-terminal to the C_(H)1 of the heavy chain or the corresponding regionof the light chain. The precise site at which the fusion is made is notcritical; particular sites are well known and may be selected in orderto optimize the biological activity, secretion, or bindingcharacteristics of the immunoadhesin.

In a preferred embodiment, the adhesin sequence is fused to theN-terminus of the Fc domain of immunoglobulin G₁ (IgG₁). It is possibleto fuse the entire heavy chain constant region to the adhesin sequence.However, more preferably, a sequence beginning in the hinge region justupstream of the papain cleavage site which defines IgG Fc chemically(i.e. residue 216, taking the first residue of heavy chain constantregion to be 114), or analogous sites of other immunoglobulins is usedin the fusion. In a particularly preferred embodiment, the adhesin aminoacid sequence is fused to (a) the hinge region and C_(H)2 and C_(H)3 or(b) the C_(H)1, hinge, C_(H)2 and C_(H)3 domains, of an IgG heavy chain.

For bispecific immunoadhesins, the immunoadhesins are assembled asmultimers, and particularly as heterodimers or heterotetramers.Generally, these assembled immunoglobulins will have known unitstructures. A basic four chain structural unit is the form in which IgG,IgD, and IgE exist. A four chain unit is repeated in the highermolecular weight immunoglobulins; IgM generally exists as a pentamer offour basic units held together by disulfide bonds. IgA globulin, andoccasionally IgG globulin, may also exist in multimeric form in serum.In the case of multimer, each of the four units may be the same ordifferent.

Various exemplary assembled immunoadhesins within the scope herein areschematically diagrammed below:

AC_(L)-AC_(L);

AC_(H)-(AC_(H), AC_(L)-AC_(H), AC_(L)-V_(H)C_(H), or V_(L)C_(L)-AC_(H));

AC_(L)-AC_(H)-(AC_(L)-AC_(H), AC_(L)-V_(H)C_(H), V_(L)C_(L)-AC_(H), orV_(L)C_(L)-V_(H)C_(H))

AC_(L)-V_(H)C_(H)-(AC_(H), or AC_(L)-V_(H)C_(H), or V_(L)C_(L)-AC_(H));

V_(L)C_(L)-AC_(H)-(AC_(L)-V_(H)C_(H), or V_(L)C_(L)-AC_(H)); and

(A-Y)_(n)-(V_(L)C_(L)-V_(H)C_(H))₂;

wherein each A represents identical or different adhesin amino acidsequences;

V_(L) is an immunoglobulin light chain variable domain;

V_(H) is an immunoglobulin heavy chain variable domain;

C_(L) is an immunoglobulin light chain constant domain;

C_(H) is an immunoglobulin heavy chain constant domain;

n is an integer greater than 1;

Y designates the residue of a covalent cross-linking agent.

In the interests of brevity, the foregoing structures only show keyfeatures; they do not indicate joining (J) or other domains of theimmunoglobulins, nor are disulfide bonds shown.

However, where such domains are required for binding activity, theyshall be constructed to be present in the ordinary locations which theyoccupy in the immunoglobulin molecules.

Alternatively, the adhesin sequences can be inserted betweenimmunoglobulin heavy chain and light chain sequences, such that animmunoglobulin comprising a chimeric heavy chain is obtained. In thisembodiment, the adhesin sequences are fused to the 3′ end of animmunoglobulin heavy chain in each arm of an immunoglobulin, eitherbetween the hinge and the C_(H)2 domain, or between the C_(H)2 andC_(H)3 domains. Similar constructs have been reported by Hoogenboom, etal., Mol. Immunol. 28:1027-1037 (1991).

Although the presence of an immunoglobulin light chain is not requiredin the immunoadhesins of the present invention, an immunoglobulin lightchain might be present either covalently associated to anadhesin-immunoglobulin heavy chain fusion polypeptide, or directly fusedto the adhesin. In the former case, DNA encoding an immunoglobulin lightchain is typically coexpressed with the DNA encoding theadhesin-immunoglobulin heavy chain fusion protein. Upon secretion, thehybrid heavy chain and the light chain will be covalently associated toprovide an immunoglobulin-like structure comprising two disulfide-linkedimmunoglobulin heavy chain-light chain pairs. Methods suitable for thepreparation of such structures are, for example, disclosed in U.S. Pat.No. 4,816,567, issued 28 Mar. 1989.

Immunoadhesins are most conveniently constructed by fusing the cDNAsequence encoding the adhesin portion in-frame to an immunoglobulin cDNAsequence. However, fusion to genomic immunoglobulin fragments can alsobe used (see, e.g. Aruffo et al., Cell 61:1303-1313 (1990); andStamenkovic et al., Cell 66:1133-1144 (1991)). The latter type of fusionrequires the presence of Ig regulatory sequences for expression. cDNAsencoding IgG heavy-chain constant regions can be isolated based onpublished sequences from cDNA libraries derived from spleen orperipheral blood lymphocytes, by hybridization or by polymerase chainreaction (PCR) techniques. The cDNAs encoding the “adhesin” and theimmunoglobulin parts of the immunoadhesin are inserted in tandem into aplasmid vector that directs efficient expression in the chosen hostcells.

Further details of the invention are provided in the followingnon-limiting Examples.

All patents, patent applications, publications, product descriptions,and protocols are cited throughout this application, the disclosures ofwhich are incorporated herein by reference in their entireties.

EXAMPLES

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC® accession numbers is the AmericanType Culture Collection, Manassas, Va.

Example 1 Production of Polypeptides in Glutamine-Free Production Medium

Materials and Methods:

Cell Lines.

In these studies, CHO host cells expressing an Apomab antibody,anti-VEGF antibody, and the fusion protein BR3-Fc, respectively wereused. The host cells were adapted in suspension and serum free cultures.Frozen stocks were prepared as master or working cell banks in the mediadescribed below.

Cell line maintenance was carried out using a 250-mL or 1-Liter Corning®vented shake flasks maintained in a Thermo Scientific Forma® reach-in aCO₂ humidified incubator maintained at 37° C. and 5% CO₂. Flasks wereagitated at rate of 150 rpm on a New Brunswick Scientific Innova®-2100platform shaker with a custom aluminum-substrate platform. Cell cultureswere passed every 3 or 4 days with fresh media and seeded at 0.11% or0.20% Packed Cell Volume (PCV). PCV was obtained using a glass 10-mLKIMAX® USA PCV tube.

Culture Media and Conditions.

Media studies were initiated using 250-mL Corning vented shake flaskinoculated in singlet, duplicate, or triplicate at 100 mL working volumeat 0.20% PCV for all cases using cell culture from a source1-LiterCorning® vented shake flask with a 500-mL working volume. PCV wasobtained using a glass 10-mL KIMAX® USA PCV tube.

Prior to initiation of the study cell culture was centrifuged at 1000rpm for 5-minutes in a Sorvall® RT 6000B centrifuge to complete a 100%media exchange of inoculum media containing glutamine with therespective test media. Different concentrations of Glutamine, Glutamate,Asparagine and Aspartate were evaluated in the different test media. Thefollowing concentrations were tested: Glutamine 0-10 mM, Glutamate 1-10mM, Asparagine 0-15 mM, Aspartate 1-10 mM. Media conditions wereevaluated in full factorial DOE studies.

The effect of Glutamine-free medium on was also tested in commerciallyavailable DMEM/F12 medium. The medium was used at 5× concentration (7.05g/L) with extra Asparagine (10 mM total), Aspartate (10 mM total),Glutamine (10 mM total for the Glutamine-containing medium), Glutamate(1 mM total), and glucose (8 g/L total). Glutamine-free andGlutamine-containing medium were compared using Apomab and anti-VEGFantibody expressing cells.

Shake flasks were maintained in a Thermo Scientific Forma® reach-in aCO₂ humidified incubator maintained at 37° C. and 5% CO₂. Flasks wereagitated at rate of 150 rpm on a New Brunswick Scientific Innova®-2100platform shaker with a custom aluminum-substrate platform.

The medium used contained the following components:

Organic Salts and Trace Elements

-   Ammonium Paramolybdate, Tetrahydrate-   Ammonium Vanadium Oxide-   Calcium Chloride, Anhydrous-   Cupric Sulfate, Pentahydrate-   Ferrous Sulfate, Heptahydrate-   Potassium Chloride-   Magnesium Chloride, Anhydrous-   Manganese Sulfate, Monohydrate-   Nickel Chloride, Hexahydrate-   Selenious Acid-   Sodium Metasilicate, Nonahydrate-   Sodium Phosphate, Monobasic, Monohydrate-   Stannous Chloride, Dihydrate-   Zinc Sulfate, Heptahydrate    Lipids-   Linoleic Acid-   Lipoic Acid (aka Thioctic Acid)-   Putrescine, Dihydrochloride    Amino Acids-   L-Alanine-   L-Arginine, Monohydrochloride-   L-Asparagine-   L-Aspartic Acid-   L-Cysteine, Monohydrochloride, Monohydrate-   L-Glutamic Acid-   L-Glutamine-   L-Histidine, Monohydrochloride, Monohydrate-   L-Isoleucine-   L-Leucine-   L-Lysine, Monohydrochloride-   L-Methionine-   L-Phenylalanine-   L-Proline-   L-Serine-   L-Threonine-   L-Tryptophan-   L-Tyrosine, Disodium Salt, Dihydrate-   L-Valine    Vitamins-   Biotin-   D-Calcium Pantothenate-   Choline Chloride-   Folic Acid-   I-Inositol-   Niacinamide-   Pyridoxine, Monohydrochloride-   Riboflavin-   Thiamine, Monohydrochloride-   Vitamin B-12    Carbon Source, Growth Factors, and Miscellaneous-   Fluronic F-68-   D-Glucose-   Sodium Bicarbonate-   Sodium Pyruvate-   Sodium Chloride-   Sodium Hydroxide-   Insulin-   Galactose

The commercially-available DMEM/F-12 culture medium was also tested,having the following components;

(mg/L) VITAMINS Biotin 0.00365 D-calcium pantothenate 2.24 Cholinechloride 8.98 Cyanocobalamin 0.68 Folic acid 2.65 i-inositol 12.6Niacinamide 2.0185 Pyridoxal HCl 2 Pyridoxine HCI 0.031 Riboflavin 0.219Thiamine HCl 2.17 AMINO ACIDS L-alanine 4.455 L-arginine HCl 147.5L-asparagine monohydrate 7.5 L-aspartic acid 6.65 L-cysteine HCImonohydrate 17.56 L-cystine 2HCl 31.29 L-glutamic acid 7.35 L-glutamine365 Glycine 18.75 L-histidine HCl monohydrate 31.48 L-isoleucine 54.47L-leucine 59.05 L-lysine HCl 91.25 L-methionine 17.24 L-phenylalanine35.48 L-proline 17.25 L-serine 26.25 L-threonine 53.45 L-tryptophan 9.02L-tyrosine 2Na dihydrate 55.79 L-valine 52.85 OTHER Dextrose anhydrous3151 HEPES 3575 Hypoxanthine sodium salt 2.39 Linoleic acid 0.042DL-α-Lipoic acid 0.105 Phenol red sodium salt 8.602 Putrescine 2HCI0.081 Sodium pyruvate 55 Thymidine 0.365 ADD: Sodium bicarbonate 1200INORGANIC SALTS Calcium chloride anhydrous 116.61 Cupric sulfatepentahydrate 0.00125 Ferric nitrate nonahydrate 0.05 Ferrous sulfateheptahydrate 0.417 Magnesium chloride anhydrous 28.61 Magnesium sulfateanhydrous 48.84 Potassium chloride 311.8 Sodium chloride 6999.5 Sodiumphosphate dibasic anhydrous 71.02 Sodium phosphate monobasic 62.5monohydrate Zinc sulfate heptahydrate 0.4315

The medium for inoculum culture (as opposed for the production phase)was usually supplemented with 5 mM glutamine, 8 g/L glucose, and 75-2000nM Methotroxate.

For studies pH adjustment was performed as needed to maintain pH valueat 7.00±0.10 using 1M Sodium Carbonate. Adjustment in pH value was madein by adding 1 mL/L of 1M Sodium Carbonate to raise pH units up 0.10.

Cell culture was analyzed up to 14-days by taking a 3.5-mL sample andanalyzed for viable cell count, viability, and cell size using a BeckmanCoulter ViCell™-1.0 cell counter. Nutrient analysis was performed usingthe Nova 400 Biomedical Bioprofile®. Osmolality was measured using anAdvanced® Instrument multi-sample Osmometer (Model 3900). Recombinantproduct titer concentration was obtained using the Agilent 1100 SeriesHPLC.

Recombinant Proteins.

The recombinant proteins produced were Apomab (TRAIL), anti-VEGF, andthe immunoadhesin BR3-Fc.

Data Analysis.

Statistical analyses of the data were carried out using a full factorialdesign of experiment, which is an experiment whose design consists oftwo or more factors, each with discrete possible values or “levels”, andwhose experimental units take on all possible combinations of theselevels across all such factors. A full factorial design may also becalled a fully-crossed design. Such an experiment allows studying theeffect of each factor on the response variable, as well as the effectsof interactions between factors on the response variable.

Results

As shown in FIGS. 1-5, use of a glutamine-free production mediumincreased the final recombinant protein titer of Apomab antibody, BR3-Fcimmunoadhesin and anti-VEGF antibody. In each case, cube plot analysisof titer results using Full Factorial DOE evaluating the effect ofdifferent concentrations of Glutamine, Glutamate, Asparagine andAspartate predict that the highest titer is achieved in Glutamine-Freemedia supplemented with 10 mM Asparagine, 10 mM Aspartic Acid and 1 mMGlutamic Acid. (FIGS. 1-3)

The effect of Asparagine under Glutamine-free, low Glutamate and highAspartate conditions on Apomab antibody titer is shown in FIG. 4. InGlutamine-free medium, Apomab antibody titer was significantly increasedin the presence of 2.5-15 mM Asparagine compared to Glutamine-freecultures without Asparagine. Under these conditions, the presence orabsence of Glutamate had no effect on titer.

Apomab antibody titer production across various Asparagine and Aspartateconcentrations in Glutamine-free and low Glutamate conditions isillustrated in FIG. 5. A positive titration effect was observed whenincreasing Aspartate from 0 to 10 mM under these conditions.

The effect of glutamine-free medium supplemented with 10 mM Asparagine,10 mM Aspartic Acid and 1 mM Glutamic Acid on titer is demonstrated inFIGS. 6A-C, wherein the final titer for Apomab antibody, anti-VEGFantibody and BR3-Fc immunoadhesin (A-C, respectively) was significantlyhigher in Glutamine-free medium compared to Glutamine-containing medium.

Similar results were obtained using the commercial DMEM/F-12 culturemedium. As shown in FIGS. 7A and B, the final titer for Apomab antibodyand anti-VEGF antibody (A and B, respectively) was significantly higherin Glutamine-free DMEM/F12 medium supplemented with 10 mM Asparagine, 10mM Aspartic Acid and 1 mM Glutamic Acid compared to Glutamine-containingDMEM F12 medium supplemented with 10 mM Asparagine, 10 mM Aspartic Acidand 1 mM Glutamic Acid.

As shown in FIGS. 8 and 9, use of a glutamine-free production mediumalso increased specific production measured as Qp (mg/mL-cell/day).FIGS. 8 A-C illustrate that cell specific productivity (Qp) for Apomabantibody, anti-VEGF antibody and BR3-Fc immunoadhesin (A-C,respectively) was significantly higher in Glutamine-free mediumsupplemented with 10 mM Asparagine, 10 mM Aspartic Acid and 1 mMGlutamic Acid compared to Glutamine-containing medium. FIGS. 9A and Billustrate that cell specific productivity for Apomab antibody andanti-VEGF antibody (A and B, respectively) was significantly higher inGlutamine-free DMEM/F12 medium supplemented with 10 mM Asparagine, 10 mMAspartic Acid and 1 mM Glutamic Acid compared to Glutamine-containingDMEM/F12 medium.

As shown in FIGS. 10 and 11, use of a glutamine-free production mediumwas shown to improve cell viability and extend culture longevitysignificantly. FIGS. 10A-C. illustrate that cell viability for Apomabantibody, anti-VEGF antibody and BR3-Fc immunoadhesin (A-C,respectively) was higher in Glutamine-free medium supplemented with 10mM Asparagine, 10 mM Aspartic Acid and 1 mM Glutamic Acid compared toGlutamine-containing medium. FIGS. 11A and B indicate that, in DMEM/F12medium, cell viability was not consistently improved in Glutamine-freemedium supplemented with 10 mM Asparagine, 10 mM Aspartic Acid and 1 mMGlutamic Acid. Of note, viability was higher for Apomab antibody (FIG.11A), but lower for anti-VEGF antibody (FIG. 11B) compared to Glutaminecontaining medium.

As shown in FIGS. 12 and 13, use of a glutamine-free production mediumreduced NH₄ ⁺ accumulation significantly compared toglutamine-containing medium. FIGS. 12 A-C illustrate that ammonia levelswere usually lower in Glutamine-free cultures supplemented with 10 mMAsparagine, 10 mM Aspartic Acid and 1 mM Glutamic Acid compared toGlutamine-containing cultures. FIGS. 13 A and B illustrate that ammonialevels were significantly reduced in Glutamine-free DMEM/F12 mediumsupplemented with 10 mM Asparagine, 10 mM Aspartic Acid and 1 mMGlutamic Acid compared to Glutamine-containing DMEM/F12 medium.

The invention illustratively described herein can suitably be practicedin the absence of any element or elements, limitation or limitationsthat is not specifically disclosed herein. Thus, for example, the terms“comprising,” “including,” “containing,” etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalent of the invention shown or portion thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modifications andvariations of the inventions embodied herein disclosed can be readilymade by those skilled in the art, and that such modifications andvariations are considered to be within the scope of the inventionsdisclosed herein.

From the description of the invention herein, it is manifest thatvarious equivalents can be used to implement the concepts of the presentinvention without departing from its scope. Moreover, while theinvention has been described with specific reference to certainembodiments, a person of ordinary skill in the art would recognize thatchanges can be made in form and detail without departing from the spiritand the scope of the invention. The described embodiments are consideredin all respects as illustrative and not restrictive. It should also beunderstood that the invention is not limited to the particularembodiments described herein, but is capable of many equivalents,rearrangements, modifications, and substitutions without departing fromthe scope of the invention. Thus, additional embodiments are within thescope of the invention and within the following claims.

All U.S. patents and applications; foreign patents and applications;scientific articles; books; and publications mentioned herein are herebyincorporated by reference in their entirety as if each individual patentor publication was specifically and individually indicated to beincorporated by reference, including any drawings, figures and tables,as though set forth in full.

What is claimed is:
 1. A process for producing a polypeptide in amammalian host cell expressing said polypeptide, comprising culturingthe mammalian host cell in a production phase of the culture in aglutamine-free production culture medium containing asparagine, whereinthe asparagine is added at a concentration in the range of 7.5 mM to 15mM.
 2. The process of claim 1 wherein the asparagine is added at aconcentration in the range of 7.5 mM to 10 mM.
 3. The process of claim 1wherein said recombinant host cell is an eukaryotic host cell.
 4. Theprocess of claim 3 wherein said eukaryotic host cell is a ChineseHamster Ovary (CHO) cell.
 5. The process of claim 4 wherein themammalian host cell is a dhfr⁻CHO cell.
 6. The process of claim 1wherein the production medium is serum-free.
 7. The process of claim 1wherein the production culture medium comprises one or more ingredientsselected from the group consisting of 1) an energy source; 2) essentialamino acids; 3) vitamins; 4) free fatty acids; and 5) trace elements. 8.The process of claim 7 wherein the production culture mediumadditionally comprises one or more ingredients selected from the groupconsisting of: 1) hormones and other growth factors; 2) salts andbuffers; and 3) nucleosides.
 9. The process of claim 1 wherein theproduction phase is a batch or fed batch culture phase.
 10. The processof claim 1 further comprising the step of isolating said polypeptide.11. The process of claim 10 further comprising determining one or moreof cell viability, culture longevity, specific productivity and finalrecombinant protein titer following isolation.
 12. The process of claim11 wherein at least one of the cell viability, culture longevity,specific productivity and final recombinant protein titer is increasedrelative to the same polypeptide produced in a glutamine-containingproduction medium of the same composition.
 13. The process of claim 1wherein the polypeptide is a mammalian glycoprotein.
 14. The process ofclaim 1 wherein the polypeptide is selected from the group consisting ofantibodies, antibody fragments, and immunoadhesins.
 15. The process ofclaim 14 wherein said antibody fragment is selected from the groupconsisting of Fab, Fab′, F(ab′)2, scFv, (scFv)2, dAb, complementaritydetermining region (CDR) fragments, linear antibodies, single-chainantibody molecules, minibodies, diabodies, and multispecific antibodiesformed from antibody fragments.
 16. The process of claim 14 wherein theantibody or antibody fragment is chimeric, humanized or human.
 17. Theprocess of claim 14 wherein said antibody or antibody fragment is atherapeutic antibody or a biologically functional fragment thereof. 18.The process of claim 17 wherein said therapeutic antibody is selectedfrom the group consisting of anti-HER2 antibodies anti-CD20 antibodies;anti-IL-8 antibodies; anti-VEGF antibodies; anti-CD40 antibodies,anti-CD11a antibodies; anti-CD18 antibodies; anti-IgE antibodies;anti-Apo-2 receptor antibodies; anti-Tissue Factor (TF) antibodies;anti-human α4β7 integrin antibodies; anti-EGFR antibodies; anti-CD3antibodies; anti-CD25 antibodies; anti-CD4 antibodies; anti-CD52antibodies; anti-Fc receptor antibodies; anti-carcinoembryonic antigen(CEA) antibodies; antibodies directed against breast epithelial cells;antibodies that bind to colon carcinoma cells; anti-CD38 antibodies;anti-CD33 antibodies; anti-CD22 antibodies; anti-EpCAM antibodies;anti-GpIIb/IIIa antibodies; anti-RSV antibodies; anti-CMV antibodies;anti-HIV antibodies; anti-hepatitis antibodies; anti-CA 125 antibodies;anti-αvβ3 antibodies; anti-human renal cell carcinoma antibodies;anti-human 17-1A antibodies; anti-human colorectal tumor antibodies;anti-human melanoma antibody R24 directed against GD3 ganglioside;anti-human squamous-cell carcinoma; and anti-human leukocyte antigen(HLA) antibodies, and anti-HLA DR antibodies.
 19. The process of claim17 wherein said therapeutic antibody is an antibody binding to a HERreceptor, VEGF, IgE, CD20, CD11a, CD40, BR3 or DR5.
 20. The process ofclaim 19 wherein said therapeutic antibody binding to DR5 is selectedfrom the group consisting of Apomabs 1.1, 2.1, 3.1, 4.1, 5.1, 5.2, 5.3,6.1, 6.2, 6.3, 7.1, 7.2, 7.3, 8.1, 8.3, 9.1, 1.2, 2.2, 3.2, 4.2, 5.2,6.2, 7.2, 8.2, 9.2, 1.3, 2.2, 3.3, 4.3, 5.3, 6.3, 7.3, 8.3, 9.3, and25.3.
 21. The process of claim 19 wherein said therapeutic antibody isan anti-BR3 antibody.
 22. The process of claim 14 wherein saidimmunadhesin is a BR3-Fc immunoadhesin.
 23. The process of claim 1wherein said polypeptide is a therapeutic polypeptide.
 24. The processof claim 23 wherein said therapeutic polypeptide is selected from thegroup consisting of a growth hormone, including human growth hormone andbovine growth hormone; growth hormone releasing factor; parathyroidhormone; thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin;insulin A-chain; insulin B-chain; proinsulin; follicle stimulatinghormone; calcitonin; luteinizing hormone; glucagon; clotting factorssuch as factor VIIIC, factor IX, tissue factor, and von Willebrandsfactor; anti-clotting factors such as Protein C; atrial natriureticfactor; lung surfactant; a plasminogen activator, such as urokinase orhuman urine or tissue-type plasminogen activator (t-PA); bombesin;thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and-beta; enkephalinase; RANTES (regulated on activation normally T-cellexpressed and secreted); human macrophage inflammatory protein(MIP-1-alpha); a serum albumin such as human serum albumin;Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; a microbial protein,such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associatedantigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelialgrowth factor (VEGF); receptors for hormones or growth factors; ProteinA or D; rheumatoid factors; a neurotrophic factor such as bone-derivedneurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4,NT-5, or NT-6), or a nerve growth factor such as NGF-β; platelet-derivedgrowth factor (PDGF); fibroblast growth factor such as aFGF and bFGF;epidermal growth factor (EGF); transforming growth factor (TGF) such asTGF-alpha and TGF-beta, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, orTGF-β5; insulin-like growth factor-I and -II (IGF-I and IGF-II);des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor bindingproteins; CD proteins such as CD3, CD4, CD8, CD19, CD20, CD34, and CD40;erythropoietin; osteoinductive factors; immunotoxins; a bonemorphogenetic protein (BMP); an interferon such as interferon-alpha,-beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF,GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxidedismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor; viral antigen such as, for example, a portion ofthe AIDS envelope; transport proteins; homing receptors; addressins;regulatory proteins; integrins such as CD11a, CD11b, CD11c, CD18, anICAM, VLA-4 and VCAM; a tumor associated antigen such as HER2, HER3 orHER4 receptor; and fragments of said polypeptides.
 25. A ready-to-useglutamine-free cell culture medium containing asparagine for theproduction of a polypeptide in a production phase, wherein theasparagine is at a concentration in the range of 7.5 mM to 15 mM.